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CN104788523A - Optimized in vivo delivery system with endosomolytic agents for nucleic acid conjugates - Google Patents

Optimized in vivo delivery system with endosomolytic agents for nucleic acid conjugates Download PDF

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CN104788523A
CN104788523A CN201510086529.4A CN201510086529A CN104788523A CN 104788523 A CN104788523 A CN 104788523A CN 201510086529 A CN201510086529 A CN 201510086529A CN 104788523 A CN104788523 A CN 104788523A
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dbait
molecule
cancer
nucleic acid
cell
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CN104788523B (en
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孙建生
马里·杜特雷克斯
马里亚·宽茨
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Valerio Treatment Co
Centre National de la Recherche Scientifique CNRS
Institut Curie
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Centre National de la Recherche Scientifique CNRS
Institut Curie
DNA Therapeutics SA
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Abstract

The present invention relates to the pharmaceutical field, and concretely relates to the field of oncology. The invention discloses an optimized in vivo delivery system with endosomolytic agents for nucleic acid of therapeutic interest conjugated to molecules facilitating endocytosis, in particular for use in the treatment of cancer.

Description

For delivery system in the body with the optimization of endosome solvating agent of nucleic conjugate
The application is international filing date is on June 21st, 2011, international application no PCT/EP2011/060280 is in the divisional application that on December 24th, 2012 enters National Phase in China, application number 201180031310.4, denomination of invention " for delivery system in the body with the optimization of endosome solvating agent of nucleic conjugate " are applied for.
Technical field
The present invention relates to pharmaceutical field, be specially oncology.
Background technology
The treatment of cancer is formed primarily of operation whenever possible, cytotoxic agent such as chemotherapy and radiotherapy.In the past decade, there is the molecule therapy for cancer therapy, such as: other kinases of signal transduction that the monoclonal antibody of targeted cells membrane receptor, the inhibitor of tyrosine kinase receptor or target participate in cell proliferation, death and survive.As cytostatic agent, their independent treatment plan often lacks enough clinical benefits.By combining with cytotoxic agent, often obtain synergetic property result, but this result is subject to the restriction of the side effect of their accumulations.
Most of cancer therapy causes DNA damage directly or indirectly in treated proliferating tumor cell, and this finally causes the death of described cell.But the tolerance that is several intrinsic and that obtain that tumour is treated these is because the effective dna repairing activity of tumour cell causes at least partly.Currently known, it is important target .Nat.Rev.Cancer such as (, 2008,8:193-204) Helleday of cancer therapy that DNA repairs.In this field, state-of-the-art drug development is PARP inhibitor.
Because DNA repairs the survival course that is absolutely necessary in all life circle, therefore, it has the reparation approach of multiple specialization, and has certain redundancy, when making when a kind of pathway deficiency or be treated agent such as DNA repair inhibitors blocking-up, this process remains sound.Therefore, not key gene/protein that target participates in DNA repair process, but, no matter its biological importance and clinical correlation, the molecule therapy of novelty by one or several critical paths property target treating in conjunction with routine treatment as a whole, thus must reach the most effective cancer therapy.
In order to make cancer can not defend existing treatment, contemplate the induction of globality target DNA damage, signal transduction and reparation approach.A kind of strategy is made up of mode below: effectively can repair DSB before importing at that time by the short dna molecule of the modification of simulated dual splitting of chain (DSB), by name Dbait and in the cell of therefore surviving.This facts explain Dbait combines the antitumor efficacy of radiotherapy (RT) or chemotherapy (CT) below: the initial DSB of Dbait molecule trapping responds to the repair signal transduction in mixture, interference downstream, disintegrate all DSB repair systems (non-homologous end joining and homologous recombination approach two kinds) subsequently, and the final DSB of suppression repairs (WO2005/040378; WO2008/034866; Quanz etc., 2009, ClinicalCancer Research 15:1308; Quanz etc., 2009, PLoS ONE 4:e6298; Dutreix etc., 2010, Mut.Res.704:182).Finally, cancer cells no longer can cheat death.Also find, when not combining with radiotherapy (RT) or chemotherapy (CT), Dbait molecule is also separately effective (WO2008/084087).
But once after having identified the promoting agent having clinical application, frequent produced problem has been to locate best mode and has carried out active agent delivery, particularly nucleic acid agent.The development of effective non-viral DNA/RNA delivery system and optimize and must solve toxicity problem, " tissue and general barrier " such as degrades, charged serum composition to the opsonization of particle, quick removing and the accumulation in non-target tissue; When by systemic routes applying active substances, for " barrier cell " that it is sent, cytoplasmic membrane is crossed in such as low picked-up, and the release of DNA molecular in active cells room is insufficient, and shortage cell nucleus targeting (required for gene therapy).
In fact, these promoting agents of great majority must be arrived tenuigenin and/or nucleus by cellular uptake, could be effectively.Particularly, when " being dissociated " with it by the promoting agent comprising nucleic acid or naked form is used, they were usually degraded before and after being absorbed by target cell.In cell, this degraded mainly causes due to this fact below: nucleic acid enters cell by endocytosis, and be isolated in the endosome of cell, endosome finally develops into lysosome, and in lysosome, chemical degradation and enzyme liberating are very effective.
In the prior art, be combined with variety carrier by promoting agent and be encapsulated in liposome, micella and nanoparticle, they are protected wherein and avoid degrading in serum.Prior art additionally uses number of chemical method and nucleic acid and other promoting agents is covalently coupled to molecular vehicle, described molecular vehicle comprises polymkeric substance such as dextran or PEG, or be intended to the molecule reducing clearance rate, comprise the carrier of Transferrins,iron complexes, lipophilic molecules is such as connected the cholesterol (Chen etc. to strengthen cellular uptake with siRNA, 2010, J.Controlled Release 144:227).Such carrier can comprise targeting moiety such as antibody, polypeptide, nucleic acid and other materials, with by promoting agent lead selected by target cell in.Prior art also discloses the molecule (US2008/0194540) for the improvement endocytosis of pharmaceutical composition.
But when active dna/RNA agent is absorbed by endocytic processes by cell, they usually finally become and are isolated in endosome that they cannot escape, significantly reduce their treatment potentiality thus.Such as, Zimmermann etc. show, the effect of cholesterol-siRNA (ApoB-1) binding substances in mouse less than its Liposomal formulation (SNALP carrier) about 1000 times: 100mg/kg chol-siApoB-1 is equivalent to the 0.1mg/kg SNALP-ApoB-1 (Nature such as Zimmermann, 2006,441:111-114, supplements Fig. 1).
For nucleic acid, prior art has been attempted by using cationic polymers such as polymine (PEI) (WO96/02655) or have the liposome that film merges lipid or peptide such as SNALP carrier and solve this problem.PEI can make endosome destabilization by the proton sponge effect clearly described, and promotes the release of nucleic acid thus.But the use of PEI is often subject to its Cytotoxic restriction, and is not up to the present also approved for the mankind.Liposomal formulation also shows toxicity and limited nucleic acid is encapsulated (usually in the scope of 1-2mg/mL), and described limited nucleic acid is encapsulated the application of the high useful load that may be not suitable for needing nucleic acid agent.
Known, " endosome dissolve (Endosomolytic) " agent such as chloroquine enters by promoting that nucleic acid is escaped from endosome the transfection that tenuigenin strengthens nucleic acid in cultured cells.But the application of chloroquine is only limitted to external application, and only send in the seldom evaluated body for auxiliary nucleic acid.Its reason may be, due to the toxicity of chloroquine, in prior art about apply in the instruction of the report of nucleic acid and its body opposing from.
Benns etc. (2000, Bioconj.Chem.11:637) report " although chloroquine has been proved to be and has contributed to plasmid DNA and discharge in tenuigenin, it is found that, chloroquine is poisonous, therefore can not use in vivo ".The partly cause of this problem is that this is true below: need the free chloroquine of rather high concentration to come the same area reaching endosome amplifying nucleic acid (i.e. plasmid DNA).Similarly, Zhang etc. (2003, J Gene Med 5:209) have studied chloroquine and apply to the body of liver for gene delivery.In this section of article, they using plasmid with use together with the peptide (polylysine/molossin) of DNA vector.Their conclusion is, although chloroquine effectively can promote that gene delivery is to liver, needs multiple dosing, and its application receives the restriction of systemic toxicity.In fact, they demonstrated that, application level in body is restricted to the level be significantly less than required for best gene delivery by the acute generalised toxicity of chloroquine.The local delivery of chloroquine is also subject to the local toxicity of chloroquine and they are from site of delivery to the restriction of external diffusion.Finally, when using naked DNA, they do not observe gene delivery or observe low-down level.
In this respect, WO2007/040469 discloses following solution: by by chloroquine and promoting agent covalent coupling, thus the total dose required for reducing, the problem of required high density chloroquine can be overcome.WO2009/126933 propose by nucleic acid to be delivered and endosome solvating agent and target part covalently bound.
Chloroquine and derivative thereof such as Oxychloroquine is used to therapeutic and the prophylactic treatment of malaria.Be investigated the radiotherapy of it and cancer and/or chemotherapeuticly combinationally use (Sotelo etc., 2006, Ann Intern Med 144:337-342; NCT01023477 and NCT00969306).Its hypothesis is, chloroquine/Oxychloroquine suppresses autophagy, and autophagy is by therapeutical agent being outputted in lysosome the normal cell defence process making them be degraded wherein.
In a word, the optimization based on the therapy of nucleic acid needs the efficiency and the cytotoxicity that solve synthetic DNA delivery system further.
Summary of the invention
The invention provides the novel effective ways of the nucleic acid for sending therepic use in body, described method be based on have the nucleic acid of therepic use with promote the covalent attachment of the molecule of endocytosis and combine have combinationally using of the nucleic acid of therepic use and endosome solvating agent.Particularly, in this body, delivery system is used to Dbait molecule.
Therefore, the present invention relates to the pharmaceutical composition of nucleic acid molecule and the quinoline endosome solvating agent comprising combination, the nucleic acid molecule of described combination has at least one free-end and has the DNA double chain portion of the 20-200bp being less than 60% sequence iden with any gene in human genome, described nucleic acid molecule with promote that the molecule covalent of endocytosis is connected, the molecule of described promotion endocytosis is selected from the part that lipophilic molecules or targeted cells acceptor make to realize receptor-mediated endocytosis.Described pharmaceutical composition can also comprise DNA damage antineoplastic agent.
The invention still further relates to product, described product comprise the nucleic acid molecule of combination and quinoline endosome solvating agent as simultaneously, separately or the combination preparation used in order, the nucleic acid molecule of described combination has at least one free-end and has the DNA double chain portion of the 20-200bp being less than 60% sequence iden with any gene in human genome, described nucleic acid molecule with promote that the molecule covalent of endocytosis is connected, the molecule of described promotion endocytosis is selected from the part that lipophilic molecules or targeted cells acceptor make to realize receptor-mediated endocytosis.Described product can also comprise DNA damage antineoplastic agent.Preferably, combine nucleic acid molecule before and/or use quinoline endosome solvating agent simultaneously.Particularly, quinoline endosome solvating agent is used at least one week as pre-treatment by oral route, then combining nucleic acid molecule and quinoline endosome solvating agent as simultaneously, separately or the combination preparation used in order use.
The present invention relates to pharmaceutical composition as disclosed herein or product, described pharmaceutical composition or product are used for the treatment of cancer.Preferably, described treatment also comprises radiotherapy or chemotherapy, preferably uses DNA damage antineoplastic agent.Optionally, DNA damage antineoplastic agent is selected from the inhibitor of the inhibitor of topoisomerase I or II, DNA linking agent, DNA alkylating agent, antimetabolite and mitotic spindle.
In a preferred embodiment, quinoline endosome solvating agent is chloroquine or Oxychloroquine, is preferably chloroquine.
In a preferred embodiment; promote that the molecule of endocytosis is selected from list or double-strand fatty acids as octadecyl and dioleoyl; tocopherol, folate or folic acid, cholesterol; sugar such as semi-lactosi and seminose and their oligosaccharides; peptide such as RGD and bombesin, and protein such as integrin, be preferably list or double-strand lipid acid, folate and cholesterol; be more preferably dioleoyl, octadecyl, folic acid and cholesterol, be also more preferably cholesterol.
In a preferred embodiment, in conjunction with nucleic acid molecule there is one of following formula:
Wherein N is Nucleotide, and n is the integer from 15 to 195, and the N of band underscore refers to the Nucleotide of the phosphodiester backbone being with or without modification, and L' is joint, and C is the molecule promoting endocytosis, and L is joint, and m is the integer for 0 or 1.Preferably, the N of underscore is with to refer to the Nucleotide of the phosphodiester backbone with modification.Preferably, the L' of connection is selected from two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of six ethylene glycol, four deoxythymidines (T4) and 2,19-; And/or m is 1 and L is formamido-oligoethylene glycol, be preferably formamido-triethylene glycol; And/or C is selected from list or double-strand lipid acid, folate and cholesterol.More preferably, the L' of connection is selected from two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of six ethylene glycol, four deoxythymidines (T4) and 2,19-; Further, m is 1 and L is formamido-oligoethylene glycol, is preferably formamido-triethylene glycol; And, C is selected from list or double-strand lipid acid, tocopherol, folate or folic acid, cholesterol, sugar such as semi-lactosi and seminose and their oligosaccharides, peptide such as RGD and bombesin and protein such as integrin, be preferably list or double-strand lipid acid, folate and cholesterol.More preferably, C is selected from dioleoyl, octadecyl, folic acid and cholesterol.Also more preferably, C is cholesterol.
In embodiment more specifically, in conjunction with nucleic acid molecule there is one of following formula:
Nucleotide wherein with underscore refers to the Nucleotide being with or without thiophosphatephosphorothioate or methylphosphonate backbone, and L' is joint, and C is the molecule promoting endocytosis, and L is joint, and m is the integer for 0 or 1.Preferably, the Nucleotide of underscore is with to refer to the Nucleotide with thiophosphatephosphorothioate or methylphosphonate backbone.Preferably, the Nucleotide of underscore is with to refer to the Nucleotide with phosphorothioate backbone; And/or the L' of connection is selected from two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of six ethylene glycol, four deoxythymidines (T4) and 2,19-; And/or m is 1 and L is oligoethylene glycol, be preferably formamido-triethylene glycol, formamido group TEG, formamido-oligoethylene glycol, be more preferably formamido-triethylene glycol; And/or, C is selected from list or double-strand lipid acid, tocopherol, folate or folic acid, cholesterol, sugar such as semi-lactosi and seminose and their oligosaccharides, peptide such as RGD and bombesin and protein such as integrin, be preferably list or double-strand lipid acid, folate and cholesterol.More preferably, the L' of connection is selected from two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of six ethylene glycol, four deoxythymidines (T4) and 2,19-; Further, m is 1 and L is formamido-polyoxyethylene glycol, is preferably formamido-triethylene glycol; Further, C is selected from list or double-strand lipid acid, folate and cholesterol.More preferably, C is selected from dioleoyl, octadecyl, folic acid and cholesterol.Also more preferably, C is cholesterol.
In very concrete embodiment, in conjunction with nucleic acid molecule be
Nucleotide wherein with underscore refers to the Nucleotide with phosphorothioate backbone.
Accompanying drawing explanation
Fig. 1: the cellular uptake of the Dbait of preparation.(A) there is the microscopical analysis (a) of the Dbait mixture of PEI11k.(B) for various transfection conditions, starting to process rear flow cytometry of carrying out cellular uptake for 5 hours.There is the Dbait-cy3 of superfect, not use before transfection and with 2 μ g/ml coDbait-cy3 of chloroquine process, not with and with the 25 μ g/ml coDbait-cy3 of CQ.
Fig. 2: the activity of the Dbait of preparation.(A) to the activation measuring DNA-PK after adding the enzyme complex of 50u purifying without DNA, 0.25 μ g Dbait or 0.25 μ g coDbait, (B) with 1.6 μ g/ml Dbait (left side), 1.6 μ g/ml Dbait/PEI11K (in), 16 μ g/mlcoDbait and latter 24 hours of CQ process, immunodetection is carried out to the γ-H2AX in cell.Scale: 20 μm.(C) by latter 5 hours of the Dbait process of various preparation (black) and 24 hours (grey), γ-H2AX's is quantitative.All transfections are all carried out with 1.6 μ g/mL Dbait or 16 μ g/mL coDbait.When pointing out, before transfection, add CQ.
Fig. 3: the phenotype being expelled to behind the space, extracellular of 1K cell stage zebrafish embryo 24 hours at Dbait.(A-C) after Dbait-cy3+PEI injection (2-5nL) to the animal pole place of 1K cell stage zebrafish embryo 24 hours, side-view anterior on the left of zebrafish embryo: top row, bright field; The end, arranges, and amplifies the head zone of 2 times, and it declines and penetrates the Dbait-cy3 of fluorescence Overlapping display redness.(A) the 1 type phenotype cannot distinguished with the (not shown) do not injected.(B) there are in head zone 2 type mild phenotype of a large amount of necrocytosis.(C) there are 3 types of strong teratogenesis and necrocytosis widely.(D) histogram shows the percentage ratio that three kinds of phenotype classifications depend on adjuvant.For often kind of condition analysis is more than the embryo of 100.NA: inject Dbait separately; Sup:Superfect; 25K, 22K, 11K PEI of corresponding size; Chloro: chloroquine; Lut:Lutrol.
Fig. 4: the diffusion in tumour and activity.With 1.6 μ g Dbait-cy5.5/PEI or 16 μ gcoDbait-cy5.5 (the unlabelled coDbait of cdDbait+9/10 of 1/10cy5.5 mark, to keep similar fluorescence intensity) inject tumour, and in next day, fluorescence distribution and DNA-PKcs activity are analyzed.After the injection of two types below, the diffusion of fluorescence Dbait a: intratumor injection or twice subcutaneous injection.
Fig. 5: 5 groups of survivals carrying the nude mice of SK28 melanoma xenograft: 1) do not treat (n=16); 2) radiation (IR, n=12); 3) radiation and peritoneal injection 1mg chloroquine (CQ, IR, n=10); 4) carry out treatment by intratumor injection 0.6mg DT01 (also referred to as CoDbait) and carry out radiation (DT01, IR, n=11) after 5h; And 5) peritoneal injection 1mg chloroquine carries out pre-treatment, intratumor injection 0.6mg DT01 (also referred to as CoDbait) after 2 hours, and carry out radiation (DT01, CQ, IR, n=13) after 5h.
Fig. 6. be transplanted to the tumor growth research of the melanoma SK28 on nude mice.Top: treatment plan: 4 kinds of DT01 (also referred to as CoDbait) of combining with 4 kinds of radiation (RT) phases in two weeks are treated.At two relative some subcutaneous injection 4mg DT01, described two relative points and tumor boundaries are separated by 5mm.Before begin treatment and at DT01+RT treatments period 1mg chloroquine (CQ), biweekly pre-treatment is carried out to animal by Orally administered (p.o.) *.Middle: the mean value of the tumor growth of various animal groups: do not treat or CQ: not treat or only with CQ process (n=11); RT or CQ+RT: have or carry out radiation (n=16) without chloroquine co-processing; DT01+RT: undertaken treating (n10) by DT01 and radiation; DT01+CQ+RT: undertaken treating (n=12) by DT01 and chloroquine and radiation.Bottom: the details of DT01+RT and DT01+CQ+RT group.Every bar curve corresponds to the growth of a tumour.
Fig. 7: active with test kit SignaTECT DNA dependent protein kinase detection system (SignaTECT DNA-dependent Protein Kinase Assay System) (Promega, Madison, WI, USA) monitoring of DNA-PK.According to the specification sheets of manufacturers, the DNA-PK (Promega, Madison, WI, USA) of biotinylated peptide substrates, 50 units and the various Dbait molecules of 500nM are hatched 5 minutes with (γ-32P) ATP at 30 DEG C.By biotinylated substrate capture on Streptavidin (streptavidin) film, washing, and count in scintillometer.By combine radioactivity divided by each sample (γ- 32p) ATP sum calculates phosphorylation percentage ratio.Dbait32Hc is unconjugated Dbait molecule.0813,0815,0902,0903,0904 and 0905 is the Dbait molecule (see " the optional Dbait molecule combined " table) combined.Dbait8H is short (8-bp) Dbait of the negative control being used as DNA-PK activity.
Fig. 8: the activity of the Dbait molecule recorded by H2AX phosphorylation.Have or without the condition of 50 μMs of chloroquine pre-treatments under latter 24 hours of the transfection Dbait molecule of various combination (see " the optional Dbait molecule combined " table), the immunodetection of γ-H2AX in MRC5 clone.The Dbait prepared by polymine (PEI) is used as positive control.
Embodiment
Import little DNA molecular (Dbait) and weaken impaired chromosomal DNA reparation, and provide the effective ways for strengthening radiotherapy or chemotherapy effect in tumour, particularly tolerance tumour.But the sensitizing activity of Dbait molecule depends on their delivery efficiencies in tumour cell.
Therefore, the present inventor compares different strategies to improve this committed step.In order to test strategy, they develop series of experiments: the analysis of molecules of the mixture that (i) is formed with Dbait molecule, (ii) for Dbait picked-up and active test cell line, (iii) zebrafish embryo Laser Scanning Confocal Microscope alive monitors distribution in vivo and the biological activity of the molecule prepared.The most effective preparation and application program is selected before these tests make it possible to analyze xenograft tumor on mouse.Compare two class preparations: have linearly or the polycationic polymer of the polymine (PEI) of branching, and the Dbait covalently bound with cholesterol (coDbait).For the in vitro and in vivo transfection of Dbait, linear PEI mixture is the most effective, but demonstrates high toxicity.In fact, compared with the Dbait prepared with PEI, need coDbait and the 1mg chloroquine of high 10 multiple doses to use together (according to allometry conversion, being equivalent to the preventive dose used in the mankind), identical anti-tumour effect could be observed in heteroplastic melanoma.But it is found that, the coDbait dosage used together with chloroquine tested is nontoxic.
Therefore, the combination of chloroquine and the chloroquine (converting animal to by allometry) of application program, particularly cholesterol-Dbait binding substances and preventive dose that the invention describes the clinical relevant dose that cholesterol-nucleic conjugate and general are used are not having the application under remarkable toxicity.The present inventor shows, in mouse, with with non-viral carrier systems as carrier 1x Dbait compared with, the cholesterol-Dbait of high 10 times amount (10x) has similar effect, instead of the amount of high 1000 times when not using chloroquine described in prior art.What this made therapeutic nucleic acids and lipotropy or cell targeting agent is combined into safety and economic available delivery system.
Therefore, although need the coDbait of more high dosage, the present inventor is surprisingly found out that:
1) if any, the combination of coDbait and chloroquine also shows low toxicity in vivo.This makes therapeutic index (ratio of effective dose/toxicity dose) can be roughly from Dbait/PEI the >20 that 1 brings up to coDbait; Inject in mouse, rat, rabbit and monkey medium sized vein, after subcutaneous injection, and even all observe after intracerebral injection and there is not toxicity;
2) combination of coDbait and chloroquine gives the DNA-PK (major target of Dbait) postponed and continue and activates, and allows to have long-term result for the treatment of.More specifically, in this time period, observe activity or the effect of increase;
3) astoundingly, compared with Dbait/PEI, coDbait is diffused in tumour/tissue well.
4) the present inventor's first observed makes the cellular uptake of coDbait increase to chloroquine, and use be combined with siRNA molecule cholesterol time, this effect is distant.
Based on these observationss, the present invention relates to
-pharmaceutical composition, it comprises the Dbait molecule of combination as described below or hairpin nucleic acid molecule and optional b) DNA damage antineoplastic agent and pharmaceutically acceptable carrier, be used for the treatment of cancer specifically;
-pharmaceutical composition, it comprises Dbait molecule or hairpin nucleic acid molecule, endosome solvating agent b) as described below, the optional c of combination a) as described below) DNA damage antineoplastic agent and pharmaceutically acceptable carrier, be used for the treatment of cancer specifically;
-product or test kit, it contains the Dbait molecule of (a) following disclosed combination or hairpin nucleic acid molecule and optional b) DNA damage antineoplastic agent as simultaneously, separately or the combination preparation used in order, specifically in the treatment of cancer;
-product or test kit, it contains Dbait molecule or hairpin nucleic acid molecule, endosome solvating agent b) as described below and the optional c of (a) following disclosed combination) DNA damage antineoplastic agent as simultaneously, separately or the combination preparation used in order, specifically in the treatment of cancer;
-combination preparation, it comprises for simultaneously, separately or the Dbait molecule of (a) that use in order following disclosed combination or hairpin nucleic acid molecule, endosome solvating agent b) as described below and optional c) DNA damage antineoplastic agent, specifically in the treatment of cancer;
-pharmaceutical composition, it comprises Dbait molecule or the hairpin nucleic acid molecule of following disclosed combination, and described pharmaceutical composition and radiotherapy and/or DNA damage antineoplastic agent combine and be used for the treatment of cancer;
-pharmaceutical composition, it comprises the Dbait molecule of a) following disclosed combination or hairpin nucleic acid molecule and endosome solvating agent b) as described below, and described pharmaceutical composition and radiotherapy and/or DNA damage antineoplastic agent combine and be used for the treatment of cancer;
-product or test kit, it contains the Dbait molecule of (a) following disclosed combination or hairpin nucleic acid molecule and endosome solvating agent b) as described below as simultaneously, separately or the combination preparation used in order, combine specifically in the treatment of cancer with radiotherapy and/or DNA damage antineoplastic agent;
-comprise the following disclosed Dbait molecule of combination or the pharmaceutical composition of hairpin nucleic acid molecule manufacturing for the medicine of radiotherapy and/or DNA damage antineoplastic agent treatment of cancer with combinations or for the medicine of effect of improving radiotherapy and/or DNA damage antineoplastic agent Therapeutic cancer or for strengthening tumour to radiotherapy and/or to the application in the medicine of the susceptibility of the treatment using DNA damage antineoplastic agent to carry out;
-comprise the following disclosed Dbait molecule of combination or the pharmaceutical composition of hairpin nucleic acid molecule manufacture for combine with radiotherapy and/or DNA damage antineoplastic agent and with following disclosed in endosome solvating agent treatment of cancer with combinations medicine in application;
-the pharmaceutical composition that comprises Dbait molecule or hairpin nucleic acid molecule and endosome solvating agent b) as described below a) combined as disclosed herein is manufacturing the application be used for the medicine of radiotherapy and/or DNA damage antineoplastic agent treatment of cancer with combinations;
-comprise Dbait molecule or hairpin nucleic acid molecule and endosome solvating agent b) as described below a) combined as disclosed herein pharmaceutical composition manufacture the effect for improving radiotherapy and/or DNA damage antineoplastic agent Therapeutic cancer medicine or for strengthening tumour to radiotherapy and/or to the application in the medicine of the susceptibility of the treatment using DNA damage antineoplastic agent to carry out;
-comprise the Dbait molecule or hairpin nucleic acid molecule, endosome solvating agent b) as described below and optional c that a) combine as disclosed herein) pharmaceutical composition of DNA damage antineoplastic agent and pharmaceutically acceptable carrier manufacturing the application be used for the treatment of in the medicine of cancer;
-for the method for Therapeutic cancer in the object needed, described method comprises the pharmaceutical composition using significant quantity, and described pharmaceutical composition comprises the Dbait molecule or hairpin nucleic acid molecule and optional DNA damage antineoplastic agent and pharmaceutically acceptable carrier that combine as disclosed herein;
-for the method for Therapeutic cancer in the object needed, described method comprises the pharmaceutical composition using significant quantity, and described pharmaceutical composition comprises the Dbait molecule or hairpin nucleic acid molecule, endosome solvating agent b) as described below and optional c that a) combine as disclosed herein) DNA damage antineoplastic agent and pharmaceutically acceptable carrier;
-for the method for Therapeutic cancer in the object needed, described method comprise use significant quantity the pharmaceutical composition comprising Dbait molecule or the hairpin nucleic acid molecule combined as disclosed herein, significant quantity comprise the pharmaceutical composition of endosome solvating agent as described below and the pharmaceutical composition comprising DNA damage antineoplastic agent of optional significant quantity;
-for the method for Therapeutic cancer in the object needed, described method comprise with radiotherapy and/or DNA damage antineoplastic agent combined administration significant quantity comprise the Dbait molecule or the pharmaceutical composition of hairpin nucleic acid molecule and the pharmaceutical composition comprising endosome solvating agent as described below of significant quantity that a) combine as disclosed herein;
-for improve in the object needed radiotherapy and/or DNA damage antineoplastic agent Therapeutic cancer effect or for strengthening the method for tumour to radiotherapy and/or the susceptibility to the treatment using DNA damage antineoplastic agent to carry out, described method comprises the pharmaceutical composition using significant quantity, and described pharmaceutical composition comprises the Dbait molecule or hairpin nucleic acid molecule, endosome solvating agent b) as described below and pharmaceutically acceptable carrier that a) combine as disclosed herein;
-for improve in the object needed radiotherapy and/or DNA damage antineoplastic agent Therapeutic cancer effect or for strengthening the method for tumour to radiotherapy and/or the susceptibility to the treatment using DNA damage antineoplastic agent to carry out, described method comprise use significant quantity comprise the Dbait molecule or the pharmaceutical composition of hairpin nucleic acid molecule and the pharmaceutical composition comprising endosome solvating agent as described below of significant quantity that combine as disclosed herein.
When using in this article, term " test kit ", " product " or " combination preparation " concrete this meaning below define " test kit of part ": namely, can independently or by using the different fixed Combination of the significant quantity of COMBINATION OF THE INVENTION (a) as defined above and (b) and optional (c), the while of i.e. or at different time points, administration is carried out to COMBINATION OF THE INVENTION (a) and (b) and optional (c).So, such as, part of the test kit using described part or can be interlocked in chronological order simultaneously, that is, part of the test kit of described part can be used with equal or not etc. the timed interval to any part of the test kit of part in different time points.The COMBINATION OF THE INVENTION (a) used with combination preparation can change with the total amount ratio of COMBINATION OF THE INVENTION (b) and optional (c).COMBINATION OF THE INVENTION (a) and (b) and optional (c) can be used by identical approach or by different approach.
In context of the present invention, term treatment represents therapeutic, symptomatic and prophylactic treatment.Pharmaceutical composition of the present invention, test kit, product and combination preparation can be used to suffer from present cancer or tumour, comprise the people in the early stage of cancer or late progression stage.Pharmaceutical composition of the present invention, test kit, product and combination preparation might not cure the patient suffering from cancer, but can postpone or delay the progress of disease or prevent further progress, thus improve the illness of patient.Particularly, pharmaceutical composition of the present invention, test kit, product and combination preparation reduce the development of tumour in mammalian hosts, reduce tumor burden, produce tumor regression, and/or prevent transfer from occurring and cancer return.In Therapeutic cancer, use pharmaceutical composition of the present invention to treat significant quantity.
" significant quantity " refer to pharmaceutical composition of the present invention in the Mammals comprising people separately or with other active ingredient combinations of pharmaceutical composition, test kit or combination preparation and prevent, eliminate or reduce the harmful effect of cancer time amount.Should be appreciated that, those skilled in the art can adjust application dosage according to patient, pathology, method of application etc.
In this whole specification sheets, " Therapeutic cancer " is mentioned for pharmaceutical composition of the present invention at every turn or similar time, just refer to: method a) being used for the treatment of cancer, the object that described method comprises to this treatment of needs uses pharmaceutical composition of the present invention; B) pharmaceutical composition of the present invention is used for the treatment of the application of cancer; C) pharmaceutical composition of the present invention is manufacturing the application be used for the treatment of in the medicine of cancer; And/or d) be used for the treatment of the pharmaceutical composition of the present invention of cancer.
dbait molecule
Dbait molecule has been widely described in PCT patent application WO2005/040378, WO2008/034866 and WO2008/084087, and the disclosure of described patent application is by reference to being incorporated into this.
Dbait molecule can be defined by many characteristics required for their therapeutic activity, such as their minimum length, there is at least one free-end, and there is double stranded section, be preferably double-stranded DNA part.As what will discuss below, importantly it should be noted that the accurate nucleotide sequence of Dbait molecule does not affect their activity.In addition, Dbait molecule can containing that modify and/or non-natural skeleton.
Preferably, Dbait molecule is nonhuman origin's (that is, their nucleotide sequence and/or conformation (such as hair clip) exist unlike in people's cell), most preferably is synthesis source.Because if some words, the sequence of Dbait molecule also plays considerably less effect, therefore, Dbait molecule preferably has sequence homology or the identity of non-significant degree with known, promotor, enhanser, 5'-or 3'-upstream sequence, exon, intron etc.In other words, any gene in Dbait molecule and human genome has the sequence iden being less than 80% or 70%, being even less than 60% or 50%.Determine that the method for sequence iden is as known in the art, and comprise such as BLASTN 2.2.25.For human genome, preferably consider that human genome structure 37 (Human Genome Build 37) (with reference to GRCh37.p2 and alternative set (alternateassembly)) is for determining identity percentage ratio.Under strict conditions, Dbait molecule not with human genomic DNA hybridization.Typical stringent condition is that they allow the nucleic acid of complete complementary and the nucleic acid of partial complementarity to distinguish such condition.
In addition, in order to avoid the receptor-mediated immune response of known toll sample, the sequence preference of Dbait molecule does not have CpG.
The length of Dbait molecule can change, as long as it is enough to the suitable combination of the Ku albumen composition allowing to comprise Ku and DNA-PKcs albumen.Show, the length of Dbait molecule must be greater than 20bp, preferably about 32bp, to guarantee be combined with such Ku mixture and allow DNA-PKcs to activate.Preferably, Dbait molecule comprises 20-200bp, more preferably 24-100bp, also more preferably 26-100, and most preferably between 32-100bp.Such as, Dbait molecule comprises 24-160,26-150,28-140,30-120 or 32-100bp." bp " refers to that this molecule comprises the double stranded section of designated length.
In a specific embodiment, the Dbait molecule with at least 32pb or about 32bp double stranded section comprises the nucleotide sequence identical with Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5).Optionally, Dbait molecule has the Nucleotide composition identical with Dbait32, Dbait32Ha, Dbait32Hb, Dbait32Hc or Dbait32Hd, but their nucleotide sequence is different.So Dbait molecule comprises a chain of the double stranded section with 3A, 6C, 12G and 11T.Preferably, the sequence of Dbait molecule is not containing any CpG dinucleotides.
Or double stranded section comprises at least 16,18,20,22,24,26,28,30 or 32 continuous nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ IDNo 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5).In embodiment more specifically, double stranded section is made up of 20,22,24,26,28,30 or 32 continuous nucleotides of Dbait32 (SEQID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5).
Dbait molecule must have the stand-in of at least one free-end as DSB.Described free-end can be free flat end or 5'-/3'-protruding terminus.In this article, " free-end " refers to nucleic acid molecule, particularly has 5' end and 3' end or has the double stranded nucleic acid segment of 3' end or 5' end.Optionally, one in 5' with 3' end can be used in conjunction with Dbait molecule or can be connected with blocking groups such as a or 3'-3' nucleotide bond.
In a specific embodiment, they contain two free-ends and can be linear.Therefore, Dbait molecule can also be the duplex molecule having two free-ends and have the nucleotide sequence of Dbait32 (SEQ ID No1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5).
In another embodiment, they are only containing a free-end.Preferably, Dbait molecule is made up of the hairpin nucleic acid with double-stranded DNA stem and ring.Described ring can be nucleic acid or other chemical groups known to the skilled or its mixture.Polynucleotide adapter can comprise 2 to 10 Nucleotide, is preferably 3,4 or 5 Nucleotide.Comprise de-nucleotide base, polyethers, polyamine, polymeric amide, peptide, carbohydrate, lipid, poly-hydrocarbon or other compounds be polymerized (such as non-nucleotide linker nonexhaustive, oligoethylene glycol, such as those have 2 to 10 ethylene glycol unit, preferably the oligoethylene glycol of 4,5,6,7 or 8 ethylene glycol unit).Preferred joint is selected from two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of six ethylene glycol, four deoxythymidines (T4) and other joints such as 2,19-.Therefore, in a specific embodiment, Dbait molecule can be the Hairpin Molecules with double stranded section or stem and ring, described double stranded section or stem comprise Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), at least 16 of Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No5), 18, 20, 22, 24, 26, 28, 30 or 32 continuous nucleotides, and described ring is six ethylene glycol joints, four deoxythymidine joints (T4) or 2, two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of 19-.In embodiment more specifically, those Dbait molecules can have the double stranded section be made up of 20,22,24,26,28,30 or 32 continuous nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5).
Dbait molecule preferably comprises 2'-deoxynucleotide backbone, and optionally comprises Nucleotide that one or several (2,3,4,5 or 6) modify and/or the core base except VITAMIN B4, cytosine(Cyt), guanine and thymus pyrimidine.Therefore, Dbait molecular nature is DNA structure.Particularly, the double stranded section of Dbait molecule or stem are made up of deoxyribonucleotide.
Preferred Dbait molecule comprises Nucleotide or the group of one or several chemically modified in the end of a chain or every bar chain, particularly in order to protect them to avoid degraded.In particularly preferred embodiments, in the end of a chain or every bar chain, the free-end of Dbait molecule by one, phosphodiester backbone that two or three are modified protects.Preferred chemical group, the phosphodiester backbone particularly modified, comprises thiophosphatephosphorothioate.Or preferred Dbait has 3'-3' nucleotide bond, or there is the Nucleotide of methylphosphonate backbone.Other skeleton modified is well known in the art, and comprise phosphoramidate, morpholino nucleic acid, 2'-O, key or short chain heteroatomic or heterocycle sugar internal key between the lock nucleic acid of 4'-C methylene radical/ethylidene bridging, peptide nucleic acid(PNA) (PNA) and short-chain alkyl or adjustable length cycloalkyl sugar, or the Nucleotide of any modification known to the skilled.In the first preferred embodiment; Dbait molecule has in the end of a chain or every bar chain by one, the free-end of phosphodiester backbone protection that two or three are modified, more preferably at least in 3' end but also more preferably simultaneously in 5' and 3' end by the free-end that the phosphodiester backbone (particularly thiophosphatephosphorothioate or methylphosphonate) that three are modified is protected.
In most preferred embodiments, Dbait molecule is hairpin nucleic acid molecule, described hairpin nucleic acid molecule comprises the DNA double chain portion of 32bp or stem (such as, have and be selected from SEQ ID No1-5, the particularly sequence of SEQ ID No 4) with the ring of two chains being connected DNA double chain portion or stem, described ring comprises and is selected from following joint or forms by being selected from following joint: six ethylene glycol, four deoxythymidines (T4) and 2, two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of 19-, the free-end of DNA double chain portion or stem (namely at the opposite place of ring) has three phosphodiester backbones (particularly connecting between phosphorothioate nucleotide) modified.
Described Dbait molecule is by chemosynthesis, half biosynthesizing or biosynthesizing, any amplification method, then obtained by any extraction and preparation method and any chemically modified.The joint provided makes to mix by standard nucleic acid chemistry synthesis.
More preferably, Dbait molecule is manufactured by specially designed convergent synthesis: along with mixing suitable tab precursor, is synthesized and prepares two complementary strands, after by their purifying, by their covalent couplings together by standard nucleic acid chemistry.
in conjunction with Dbait molecule
The present invention relates to and the Dbait molecule promoting that the molecule of endocytosis or cellular uptake is combined.
Particularly, described molecule can be lipophilic molecules, such as cholesterol, list or double-strand lipid acid; Or targeted cells acceptor makes the part realizing receptor-mediated endocytosis, such as folic acid and folate derivatives or Transferrins,iron complexes (the Ann.Rev.Cell Biol.19851:1-39 such as Goldstein; Leamon & Lowe, Proc Natl Acad Sci USA.1991,88:5572 – 5576.).Lipid acid can be saturated or undersaturated, and can be C 4-C 28, be preferably C 14-C 22, be also more preferably C 18, such as oleic acid or stearic acid.Particularly, lipid acid can be octadecyl or dioleoyl.Find, lipid acid can be the double chain form connected with suitable joint such as glycerine, phosphatidylcholine or thanomin etc., or can be the double chain form linked together by the joint for being connected on Dbait molecule.When using in this article, the meaning of term " folate " refers to folate and folate derivatives, comprises pteroic acid derivative and analogue.Be applicable to folacin of the present invention and derivative includes but not limited to antifol, dihydrofolic acid salt, tetrahydrofolate, folinic acid, VitB11,1-denitrogenation, 3-denitrogenation, 5-denitrogenation, 8-denitrogenation, 10-denitrogenation 1,5-denitrogenation, 5,10-bis-denitrogenation, 8,10-bis-denitrogenations and 5,8-bis-denitrogenation folate, antifol, and pteroic acid derivative.Other folate analogues are described in US2004/242582.Promote that the molecule of endocytosis can be tocopherol, sugar such as semi-lactosi and seminose and their oligosaccharides, peptide such as RGD and bombesin and protein such as integrin.Therefore, promote that the molecule of endocytosis can be selected from list or double-strand lipid acid, folate and cholesterol.More preferably, promote that the molecule of endocytosis is selected from dioleoyl, octadecyl, folic acid and cholesterol.In most preferred embodiments, Dbait molecule is combined with cholesterol.
Promote that the molecule of endocytosis is combined with Dbait molecule preferably by joint.Any joint as known in the art may be used to promote that the molecule of endocytosis is connected with Dbait molecule covalent.Such as, WO09/126933 provides wide in range property summary about joint easily at 38-45 page.Nonexhaustive ground, joint can be aliphatic chain, polyethers, polyamine, polymeric amide, peptide, carbohydrate, lipid, poly-hydrocarbon, or the compound of other polymerizations (such as, oligoethylene glycol, such as those have 2 to 10 ethylene glycol unit, preferably 3, 4, 5, 6, 7 or 8 ethylene glycol unit, the also oligoethylene glycol of more preferably 6 ethylene glycol unit), and be mixed with any key that can be cut off by chemistry or enzymolysis process, such as disulfide linkage, shielded disulfide linkage, the key (such as hydrazone key) that acid is unstable, ester bond, original acid ester key, phosphonic amide key, can the biological peptide bond sheared, azo bond or aldehyde key.This joint sheared refers to WO2007/040469 12-14 page, WO2008/022309 22-28 page.
In a specific embodiment, the Dbait molecule point sub-connection that can promote endocytosis with.Or, sub-connection can be divided the molecule of several promotion endocytosis (such as two, three or four) with a Dbait.
In specific embodiment, the joint between the molecule, the particularly cholesterol that promote endocytosis and Dbait molecule is CO-NH-(CH 2-CH 2-O) n, wherein n is the integer from 1 to 10, and preferred n is selected from 3,4,5 and 6.In very concrete embodiment, joint is CO-NH-(CH 2-CH 2-O) 4(formamido-triethylene glycol).Joint can in office what easily and do not change position and the Dbait point of sub-connection of Dbait molecular activity.Particularly, in 5' end, at 3' end jointing, or when Dbait molecule is hair clip, joint can be connected in ring.But when hair clip Dbait molecule, the present inventor is surprised to find, in the 5' end of Dbait molecule by the cholesterol of joint and Dbait point of sub-connection than dividing the cholesterol of sub-connection more effective at ring place by joint and Dbait.Therefore, in a preferred embodiment, the Dbait molecule of contemplated combination be there is hairpin structure and preferably in its 5' end by the Dbait molecule that joint is combined with the molecule of promotion endocytosis.
In another embodiment, the joint between the molecule, the particularly cholesterol that promote endocytosis and Dbait molecule is dialkyl disulphides { such as, (CH 2) p-S-S-(CH 2) q, wherein p and q is from 1 to 10, preferably from the integer of 3 to 8, such as 6 }.
In most preferred embodiments, in conjunction with Dbait molecule be hairpin nucleic acid molecule, described hairpin nucleic acid molecule comprises the DNA double chain portion of 32bp or stem (such as, have and be selected from SEQID No 1-5, the particularly sequence of SEQ ID No 4) with the ring of two chains being connected DNA double chain portion or stem, described ring comprises and is selected from following joint or forms by being selected from following joint: six ethylene glycol, four deoxythymidines (T4) and 2, two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of 19-, the free-end of DNA double chain portion or stem (namely at the opposite place of ring) has three phosphodiester backbones (particularly connecting between phosphorothioate nucleotide) modified, and described Dbait molecule is preferably by joint (such as formamido-oligoethylene glycol, preferred formamido-triethylene glycol) be combined with cholesterol in its 5' end.
In conjunction with Dbait molecule or hairpin nucleic acid molecule also can be described by following formula:
Wherein N is Nucleotide, n be greater than 14 integer, the N of band underscore refers to the Nucleotide of the phosphodiester backbone being with or without modification, and L' is joint, and C is the molecule promoting endocytosis, and L is joint, and m is the integer for 0 or 1.Preferably, the N of underscore is with to refer to the Nucleotide of the phosphodiester backbone with modification.In formula (II) and (III), C-L mbe connected with the 5' end of Nucleotide or 3' end separately.In formula (I-III), C-L mbe connected with L' preferably by disulfide linkage (S-S).
In a preferred embodiment, the molecule of formula (I), (II) or (III) has the feature below or several:
-N is deoxynucleotide, be preferably selected from A (VITAMIN B4), C (cytosine(Cyt)), thymus pyrimidine (T) and G (guanine), and be selected such that to avoid occurring CpG dinucleotides and there is with any gene in human genome the sequence iden that is less than 80% or 70%, is even less than 60% or 50%; And/or,
-n be from 15 to 195, preferably from 19-95, more preferably from 21 to 95, also more preferably from 27 to 95 integer.In particularly preferred embodiments, n is 27; And/or,
The N of-band underscore refers to the Nucleotide having thiophosphatephosphorothioate or methylphosphonate backbone, be more preferably phosphorothioate backbone; And/or,
The L' of-connection is selected from two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of six ethylene glycol, four deoxythymidines (T4) and 2,19-; And/or,
-m is 1 and L is formamido-oligoethylene glycol, is more preferably formamido-triethylene glycol; And/or,
-C is selected from cholesterol, list or double-strand fatty acids part (comprising peptide, protein, aptamer (aptamer)) such as folate and Transferrins,iron complexes as oleic acid or stearic acid or targeted cells acceptor; be preferably cholesterol, octadecyl, dioleoyl or folate, be more preferably cholesterol.
Preferably, C-Lm is triethylene glycol joint (10-O-[1-propyl group-3-N-formamyl cholesteryl]-triethylene glycol group.
In a preferred embodiment, in conjunction with Dbait molecule or hairpin nucleic acid molecule there is following formula:
Wherein identical with (III) to N, band N, n, L, L', C of underscore and the definition of m and formula (I), (II).
In a preferred embodiment, nNNn-(N) n-N comprises Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), at least 16 of Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5), 18, 20, 22, 24, 26, 28, 30 or 32 continuous nucleotides, or by Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), 20 of Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5), 22, 24, 26, 28, 30 or 32 continuous nucleotide compositions.In a specific embodiment, nNNn-(N) n-N comprises Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5), be more preferably Dbait32Hc (SEQ ID No 4), or by Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5), be more preferably Dbait32Hc (SEQ ID No 4) composition.
Therefore, in conjunction with Dbait molecule or hairpin nucleic acid molecule can be selected from:
Wherein nNNn-(N) n-N is SEQ ID No 1
Wherein nNNn-(N) n-N is SEQ ID No 2
Wherein nNNn-(N) n-N is SEQ ID No 3
Wherein nNNn-(N) n-N is SEQ ID No 4
Wherein nNNn-(N) n-N is SEQ ID No 5
Wherein identical with (III) to the definition of L, L', C and m and formula (I), (II).
In a preferred embodiment, the molecule of formula (Ia), (IIa), (IIIa), (Ib), (IIb), (IIIb), (Ic), (IIc), (IIIc), (Id), (IId), (IIId), (Ie), (IIe) and (IIIe), the molecule of preferred formula (II), (IIa), (IIb), (IIc), (IId) and (IIe), has the feature below or several:
The Nucleotide of-band underscore refers to the Nucleotide being with or without thiophosphatephosphorothioate or methylphosphonate backbone, is more preferably the Nucleotide with thiophosphatephosphorothioate or methylphosphonate backbone, is also more preferably the Nucleotide with phosphorothioate backbone; And/or,
The L' of-connection is selected from two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of six ethylene glycol, four deoxythymidines (T4) and 2,19-; And/or,
-m is 1 and L is formamido-oligoethylene glycol, is more preferably formamido-triethylene glycol; And/or,
-C is selected from cholesterol, list or double-strand fatty acids part (comprising peptide, protein, aptamer) such as folate, Transferrins,iron complexes as oleic acid or stearic acid or targeted cells acceptor; be preferably cholesterol, octadecyl, dioleoyl or folate, be more preferably cholesterol.
Preferably, C-Lm is triethylene glycol joint (10-O-[1-propyl group-3-N-formamyl cholesteryl]-triethylene glycol group.
At formula (I), (II), (III), (Ia), (IIa), (IIIa), (Ib), (IIb), (IIIb), (Ic), (IIc), (IIIc), (Id), (IId), (IIId), (Ie), and (IIIe) (IIe), be preferably formula (II), (IIa), (IIb), (IIc), (IId) and in the Dbait molecule of (IIe) or the embodiment of hairpin nucleic acid molecule, L' is preferably selected from six ethylene glycol, four deoxythymidines (T4) and 2, two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of 19-.
At formula (I), (II), (III), (Ia), (IIa), (IIIa), (Ib), (IIb), (IIIb), (Ic), (IIc), (IIIc), (Id), (IId), (IIId), (Ie), (IIe) and (IIIe), be preferably in formula (II), (IIa), (IIb), (IIc), the Dbait molecule of (IId) and (IIe) or the embodiment of hairpin nucleic acid molecule, wherein C is cholesterol, C-L mit is group
In a preferred embodiment, in conjunction with Dbait molecule or hairpin nucleic acid molecule be selected from (II), (IIa), (IIb), (IIc), (IId) and (IIe), wherein C-L mit is group
And wherein L' is preferably selected from six ethylene glycol, four deoxythymidines (T4) and 2, two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of 19-, be more preferably two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of 2,19-.
In very concrete embodiment, Dbait molecule or hairpin nucleic acid molecule have following formula
Wherein C-L mit is group
Wherein L' is two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of 2,19-, and the Nucleotide wherein with underscore has phosphorothioate backbone.Therefore, this molecule has structure below, and it is referred to as " coDbait " in embodiment part.
A kind of cholesterol-Dbait binding substances of DT01 by name is by by 1, the oligodeoxyribonucleotide of the 64-nt that two 32-nt chains with complementary sequence that two (the phosphorus)-8-hydraza-2-hydroxyl-4-oxa--9-oxo-nonadecane joint of 19-connects form, the oligodeoxyribonucleotide of described 64-nt has cholesteryl TEG in 5' end and respectively has key between 3 phosphorothioate nucleotide in 5' and 3' end.In the solution, this molecule forms hair clip 32-bp duplex in molecule.This double-strand (ds) DNA structure is absolutely necessary for its biological activity, and is active pharmaceutical ingredient (API).The molecular formula of sodium salt: C 678h 820n 244na 65o 392p 65s 6; The molecular weight of sodium salt: 22359.2Da; The molecular weight of free acid: 20931.4Da.This molecule also can be expressed as followsin:
For the molecule of formula (II), (IIa), (IIb), (IIc), (IId) or (IIe), a very surprising aspect of the present invention is: although the activity of Dbait molecule needs to there is at least one free-end, the molecule of the promotion endocytosis be connected with 5' end does not reduce activity.
Therefore, the invention still further relates to the Dbait molecule combined as disclosed above; Pharmaceutical composition, it comprises the Dbait molecule of described combination and optional pharmaceutically acceptable carrier; The Dbait molecule of combination as disclosed above, it is separately or combine with the chemotherapy of radiotherapy and/or DNA damage antineoplastic agent and be used for the treatment of cancer; Be used for the treatment of the method for cancer, it comprises the Dbait molecule of the combination as disclosed above of administering therapeutic significant quantity; And the as disclosed above Dbait molecule combined is for the preparation of the application in the medicine of Therapeutic cancer, details are as follows.
endosome solvating agent
Herein, in conjunction with Dbait molecule or hairpin nucleic acid molecule preferably combinationally use with endosome solvating agent (such as chloroquine, film merge lipid or peptide etc.).In fact, the treatment undertaken by endosome solvating agent promotes that the Dbait molecule combined discharges from endosome.In addition, this is the further wonderful effect of combination permission acquisition specifically, the result that the activity comprising low toxicity in vivo and delay and lasting Dbait mediation makes an exception like this.
Particularly, endosome solvating agent can realize dissolving endosome and can wrap up being encapsulated or wrapping up composition of therapeutical agent to be delivered to cell or subcellular component in response to the change of pH.Endosome dissolved substance includes but not limited to quinoline compound, particularly 4-quinolylamine and 2-phenylquinoline compound and amino, sulfo-, phenyl, alkyl, vinyl and halogen derivative, and film merges lipid, peptide or protein.
In a preferred embodiment, endosome solvating agent is small molecules.Alkalescence endosome solvating agent can be selected from quinine, chloroquine, Oxychloroquine, amodiaquine (Miaquin), amopyroquine, primaquine, Mefloquine hydrochloride, nivaquines, halofantrine, quinonimine and combination thereof.Preferred endosome solvating agent is quinoline endosome solvating agent, includes but not limited to be listed in compound below with its chemical name: the chloro-4-of 7-(4-diethylin-1-methyl butyl-amino) quinoline (chloroquine), the chloro-4-of 7-(4-ethyl-(2-hydroxyethyl)-amino-1-methyl butyl-amino) quinoline (Oxychloroquine), the fluoro-4-of 7-(4-diethylin-1-methyl butyl-amino) quinoline, 4-(4-diethylin-1-Methylbutylamino) quinoline, 7-hydroxyl-4-(4-diethyl-amino-1-Methylbutylamino) quinoline, the chloro-4-of 7-(4-diethylin-1-butyl is amino) quinoline (demethylation chloroquine), the fluoro-4-of 7-(4-diethylin-1-butyl is amino) quinoline), 4-(4-diethyl-amino-1-butyl is amino) quinoline, 7-hydroxyl-4-(4-diethylin-1-butyl is amino) quinoline, the chloro-4-of 7-(1-carboxyl-4-diethylin-1-butyl is amino) quinoline, the fluoro-4-of 7-(1-carboxyl-4-diethyl-amino-1-butyl is amino) quinoline, 4-(1-carboxyl-4-diethylin-1-butyl is amino) quinoline, 7-hydroxyl-4-(1-carboxyl-4-diethylin-1-butyl is amino) quinoline, the chloro-4-of 7-(1-carboxyl-4-diethylin-1-Methylbutylamino) quinoline, the fluoro-4-of 7-(1-carboxyl-4-diethyl-amino-1-Methylbutylamino) quinoline, 4-(1-carboxyl-4-diethylin-1-Methylbutylamino) quinoline, 7-hydroxyl-4-(1-carboxyl-4-diethylin-1-Methylbutylamino) quinoline, the fluoro-4-of 7-(4-ethyl-(2-hydroxyethyl)-amino-1-Methylbutylamino) quinoline, 4-(4-ethyl-(2-hydroxy-ethyl)-amino-1-Methylbutylamino-) quinoline, 7-hydroxyl-4-(4-ethyl-(2-hydroxyethyl)-amino-1-Methylbutylamino) quinoline, Oxychloroquine phosphoric acid salt, the chloro-4-of 7-(4-ethyl-(2-hydroxyethyl-l)-amino-1-butyl is amino) quinoline (demethylation Oxychloroquine), the fluoro-4-of 7-(4-ethyl-(2-hydroxyethyl)-amino-1-butyl is amino) quinoline, 4-(4-ethyl-(2-hydroxyethyl)-amino-1-butyl is amino) quinoline, 7-hydroxyl-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butyl is amino) quinoline, the chloro-4-of 7-(1-carboxyl-4-ethyl-(2-hydroxyethyl)-amino-1-butyl is amino) quinoline, the fluoro-4-of 7-(1-carboxyl-4-ethyl-(2-hydroxyethyl)-amino-1-butyl is amino) quinoline, 4-(1-carboxyl-4-ethyl-(2-hydroxyethyl)-amino-1-butyl is amino) quinoline, 7-hydroxyl-4-(1-carboxyl-4-ethyl-(2-hydroxyethyl)-amino-1-butyl is amino) quinoline, the chloro-4-of 7-(1-carboxyl-4-ethyl-(2-hydroxyethyl)-amino-1-Methylbutylamino) quinoline, the fluoro-4-of 7-(1-carboxyl-4-ethyl-(2-hydroxyethyl)-amino-1-Methylbutylamino) quinoline, 4-(1-carboxyl-4-ethyl-(2-hydroxyethyl)-amino-1-Methylbutylamino) quinoline, 7-hydroxyl-4-(1-carboxyl-4-ethyl-(2-hydroxyethyl)-amino-1-Methylbutylamino) quinoline, 8-[(4-Aminopentyl) amino-6-methoxy quinoline dihydrochloride, 1-ethanoyl-1,2,3,4-tetrahydroquinoline, 8-[(4-Aminopentyl) is amino]-6-methoxy quinoline dihydrochloride, 1-butyryl radicals-1,2,3,4-tetrahydroquinoline, the chloro-4-of 3-(4-hydroxyl-alpha, α '-bis-(2-methyl isophthalic acid-pyrrolidyl)-2,5-xylidino quinoline, 4-[(4-diethyl-amino)-1-methyl butyl-amino]-6-methoxy quinoline, the fluoro-4-of 3-(4-hydroxyl-alpha, α '-bis-(2-methyl isophthalic acid-pyrrolidyl)-2,5-xylidino quinoline, 4-[(4-diethylin)-1-methyl butyl-amino]-6-methoxy quinoline, 4-(4-hydroxyl-alpha, α '-bis-(2-methyl isophthalic acid-pyrrolidyl)-2,5-xylidino quinoline, 4-[(4-diethylin)-1-methyl butyl-amino]-6-methoxy quinoline, 3,4-dihydro-1-(2H)-quinoline carboxylic aldehyde, 1,1'-pentamethylene two quinoline diiodide, oxine vitriol, and amino, aldehyde, carboxyl, hydroxyl, halogen, ketone, sulfydryl and ethenyl derivatives or analogue.Other reagent are disclosed in (1997, J Pharmacol Exp Therapy 280:884-893) such as Naisbitt and US 5,736,557.In preferred embodiment, endosome solvating agent can be selected from chloroquine, Oxychloroquine, demethylation chloroquine, Oxychloroquine phosphoric acid salt and demethylation Oxychloroquine, is preferably chloroquine or Oxychloroquine, is more preferably chloroquine.
In another embodiment, endosome solvating agent is that film merges lipid, peptide or protein.In fact, many films merge lipid, peptide or protein is as known in the art.Such as, film merge lipid, peptide or protein be disclosed in following patent application those: WO10057160, US2007/0293449, US2006/0051405, WO10053489, WO09126933.Particularly, WO09/126933 provides film fusion lipid, peptides and proteins at 23-29 page.
The present inventor confirms the high resistance tumor therapeutic efficacy of the Dbait molecule of combination and the combination of chloroquine, but they observe, and the chloroquine of identical amount is independent or do not show any anti-tumor activity with radiating composite.
Therefore, the present invention relates to pharmaceutical composition, it comprises the Dbait molecule of combination of the present invention or hairpin nucleic acid molecule and endosome solvating agent, is used for the treatment of cancer more specifically.The invention still further relates to product, it comprises the Dbait molecule of combination of the present invention or hairpin nucleic acid molecule and endosome solvating agent as simultaneously, separately or the combination preparation used in order, be used for the treatment of cancer more specifically.Preferably, endosome solvating agent is selected from chloroquine or Oxychloroquine, is also more preferably chloroquine.Preferably, the Dbait molecule of combination of the present invention or hairpin nucleic acid molecule are the Dbait molecules of any concrete combination as above.In one embodiment, Dbait molecule of the present invention or hairpin nucleic acid molecule are covalently attached to endosome solvating agent, are preferably chloroquine or Oxychloroquine, are also more preferably chloroquine, specifically as disclosed in WO2007/040469.Another preferred embodiment in, endosome solvating agent, is preferably chloroquine and does not combine (that is, not covalently bound) with the Dbait molecule of combination of the present invention or hairpin nucleic acid molecule.
Besides the active ingredients, contemplated herein pharmaceutical composition can also comprise pharmaceutically acceptable carrier.The bioactive validity being meant to forgive not interferon activity composition of term " pharmaceutically acceptable carrier " and virose any carrier (such as upholder, material, solvent etc.) is not had to the host that it is used.Such as, parenteral is used, can medium such as salt solution, glucose solution, serum albumin and Ringer's solution active compound be formulated in injection unit dosage form.
Pharmaceutical composition can be mixed with the solution in drug compatibility solvent in a manner known in the art, or be mixed with the emulsion in suitable drug solvent or medium, suspension or dispersion, or be mixed with pill, tablet or the capsule containing solid dielectric.It is of the present invention that to be suitable for Orally administered preparation can be discrete unit form, as capsule, bag agent, tablet or lozenge, and the activeconstituents respectively containing predetermined amount; Powder or granular form; Solution in waterborne liquid or non-aqueous liquid or form of suspension; Or emulsion oil-in-water or water-in-oil emulsion form.Be suitable for aseptic oiliness or aqueous formulation that preparation that parenteral uses comprises activeconstituents easily, it is preferably isotonic with the blood of recipient.Each such preparation can also contain other drug consistency and nontoxic auxiliary, such as stablizer, antioxidant, tackiness agent, dyestuff, emulsifying agent or flavoring substance.Preparation of the present invention comprises activeconstituents and pharmaceutically acceptable carrier, therefore and optionally also has other therapeutic ingredient.Carrier is from compatible with other composition of preparation and must be " acceptable " concerning the harmless meaning of its recipient.Under vantage, carry out drug application composition by the sterile solution injected or intravenous infusion is suitable, or as oral dosage, carry out drug application composition by digestive tube.Safety and the effective application process of these chemotherapeutics of major part are well known by persons skilled in the art.In addition, in normative document, using of they is described.
Pharmaceutical composition of the present invention is not liposome composition.Particularly, the Dbait molecule of the combination of product of the present invention is not formulated in liposome composition.
Particularly, the invention still further relates to product, test kit or combination preparation, it comprises Dbait molecule as was disclosed earlier or the hairpin nucleic acid molecule of (a) one or more unit dosage form, the endosome solvating agent as was disclosed earlier of (b) one or more unit dosage form, and the following disclosed DNA damage antineoplastic agent of optional (c) one or more unit dosage form.
dNA damage is treated
Except combine Dbait molecule and endosome solvating agent except, treatment can further include antineoplaston, the treatment carried out preferably by DNA damage agent or radiotherapy.The chemotherapy that DNA damage treatment can be radiotherapy or use DNA damage antineoplastic agent to carry out, or its combination.
DNA splitting of chain can be realized by ionizing rays (radiotherapy).Radiotherapy includes, but are not limited to gamma-radiation, X-ray and/or radio isotope targeted delivery to tumour cell.Other radiotherapy comprise microwave and UV-radiation.Radiotherapeutic additive method is also contemplated in the present invention.
DNA damage antineoplastic agent is preferably selected from the inhibitor of the inhibitor of topoisomerase I or II, DNA linking agent, DNA alkylating agent, antimetabolite and mitotic spindle.
The inhibitor of topoisomerase I and/or II includes but not limited to Etoposide, and open up pool for health, camptothecine, irinotecan, amsacrine, intoplicine, anthracycline is Dx, epirubicin, daunorubicin, idarubicin and mitoxantrone such as.The inhibitor of topoisomerase I and II includes but not limited to intoplecin.
DNA linking agent includes but not limited to cis-platinum, carboplatin and oxaliplatin.
Antimetabolite blocks the enzyme being responsible for nucleic acid synthesis, or becomes and be incorporated in DNA, this generates incorrect genetic code and causes apoptosis.Its nonexhaustive example includes but not limited to antifol, pyrimidine analogue, purine analogue and adenosine deaminase inhibitors, be more specifically Rheumatrex, floxuridine, cytosine arabinoside, Ismipur, 6-Tioguanine, fludarabine phosphate, pentostatin, 5 FU 5 fluorouracil, gemcitabine and capecitabine.
DNA damage antineoplastic agent can be alkylating agent, includes but not limited to mustargen, aziridine derivative, alkyl sulfonic ester, nitrosourea, metal-salt and triazene.Its nonexhaustive example comprises uracil mustard, methine chlorine, endoxan (CYTOXAN (R)), ifosfamide, melphalan, Chlorambucil, pipobroman, three ethylene melamines, TESPA (Triethylenethiophosphoramine), busulfan, carmustine, lomustine, fotemustine, cis-platinum, carboplatin, oxaliplatin, phosphinothioylidynetrisaziridine, U-9889, Dacarbazine and Temozolomide.
The inhibitor of mitotic spindle includes but not limited to taxol, Docetaxel, vinorelbine, and larotaxel is (also referred to as XRP9881; Sanofi-Aventis), XRP6258 (Sanofi-Aventis), BMS-184476 (Bristol-Meyer-Squibb), BMS-188797 (Bristol-Meyer-Squibb), BMS-275183 (Bristol-Meyer-Squibb), Ortataxel are (also referred to as IDN 5109, BAY 59-8862 or SB-T-101131; Bristol-Meyer-Squibb), RPR 109881A (Bristol-Meyer-Squibb), RPR116258 (Bristol-Meyer-Squibb), NBT-287 (TAPESTRY), PG-taxol are (also referred to as CT-2103, PPX, PPX, polyglutamic acid taxol or Xyotax tM), (also referred to as Nab-Paclitaxel; ABRAXISBIOSCIENCE), Tesetaxel (also referred to as DJ-927), IDN 5390 (INDENA), Taxoprexin are (also referred to as the sour taxol of 22 carbon six; PROTARGA), DHA-taxol (also referred to as ) and MAC-321 (WYETH).Also can see the summary of Hennenfent & Govindan (2006, Annals of Oncology, 17,735-749).
cancer to be treated or tumour
Pharmaceutical composition described in the present invention and product, test kit or combination preparation can be used for the cancer in treatment target.
Term " cancer ", " carcinous " or " pernicious " refer to or describe characteristic feature in Mammals and be the physiological condition of not controlled Growth of Cells.The example of cancer comprises such as leukemia, lymphoma, blastoma, cancer and sarcoma.The example more specifically of this cancer comprises chronic myelogenous leukemia, acute lymphoblastic leukemia, Philadelphia Chromosome Positive acute lymphoblastic leukemia (Ph+ALL), squamous cell carcinoma, small cell lung cancer, nonsmall-cell lung cancer, glioma, gastrointestinal cancer, kidney, ovarian cancer, liver cancer, colorectal cancer, carcinoma of endometrium, kidney, prostate cancer, thyroid carcinoma, neuroblastoma, carcinoma of the pancreas, glioblastoma multiforme, cervical cancer, cancer of the stomach, bladder cancer, hepatocellular carcinoma, mammary cancer, colorectal carcinoma, and head and neck cancer, cancer of the stomach, blastoma, pediatric sarcomas, nose type natural killer cell, multiple myeloma, acute myeloid leukaemia (AML), lymphocytic leukemia, mastocytosis and any symptom relevant to mastocytosis.
" leukemia " refers to the progressivity malignant disease of hemocytopoietic organ, and general feature is propagation and the growth of the distortion of white corpuscle in blood and marrow and precursor thereof.Leukemia is usual clinically classifies according to these points: the time length of (1) disease and characteristic---and acute or chronic; (2) cell type involved by: marrow (myeloide), lymphocytic (lymphogenous) or monocarpotic cellularity; And paracytic quantity increases or does not increase in (3) blood---white courage and uprightness or non-white courageous and upright (sub-white courageous and upright).Leukemia comprises such as acute nonlymphocytic leukemia, lymphocytic leukemia, acute myeloblastic leukemia, chronic myelocytic leukemia, acute promyelocytic leukemia, adult T cell leukemia, aleukemic leukemia, the hypophosphatemic leukemia of white cell (leucocythemic leukemia), Basophilic leukemia, stem cell leukemia (blast cell leukemia), bovine leucosis, chronic granulocytic leukemia, leukemia cutis, Embryo leukemia, acidophilia leukemia, gross' leukemia, hairy cell leukemia, hemoblastic leukemia (hemoblastic leukemia), hemoblastic leukemia (hemocytoblastic leukemia), histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphoid leukemia, lymphoblastic leukemia, Lymphocytic leukemia, become lymphoid leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, Micromyeloblast leukemia, monocytic leukemia, myeloblastosis, myelocytic leukemia, marrow myelocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, Plasmacytic leukemia, plasma cell leukemia, promyelocytic leukemia, rieder's cell leukemia, schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.In some aspects, the invention provides the treatment for chronic myelogenous leukemia, acute lymphoblastic leukemia and/or Philadelphia Chromosome Positive acute lymphoblastic leukemia (Ph+ALL).
Scope of the present invention also forgives various cancer, include but not limited to below these: cancer, comprise bladder cancer (comprising acceleration and transitivity bladder cancer), mammary cancer, colorectal carcinoma (comprising colorectal cancer), kidney, liver cancer, lung cancer (comprising minicell and nonsmall-cell lung cancer and adenocarcinoma of lung), ovarian cancer, prostate cancer, carcinoma of testis, genitourinary cancer, lymphsystem cancer, the rectum cancer, laryngocarcinoma, carcinoma of the pancreas (comprising exocrine pancreas cancer), esophagus cancer, cancer of the stomach, carcinoma of gallbladder, cervical cancer, thyroid carcinoma and skin carcinoma (comprising squamous cell carcinoma), lymphatic cells system tumor, comprises leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B cell lymphoma, t cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma, histocytic lymphoma and Burketts lymphoma, medullary system hematopoietic system cancer, comprises acute and chronic myelogenous leukemia, myelodysplastic syndrome, myelogenous leukemia and promyelocytic leukemia, the tumour of maincenter and peripheral nervous system, comprises astrocytoma, neuroblastoma, glioma and schwannoma, the tumour of mesenchyme origin, comprises fibrosarcoma, rhabdosarcoma and osteosarcoma, other tumours, comprise melanoma, xeroderma pitmentosum, keratoacanthoma, spermocytoma, follicular carcinoma of thyroid and teratoma, melanoma, can not III phase of excision or IV phase malignant melanoma, squamous cell carcinoma, small cell lung cancer, nonsmall-cell lung cancer, glioma, gastrointestinal cancer, kidney, ovarian cancer, liver cancer, colorectal cancer, carcinoma of endometrium, kidney, prostate cancer, thyroid carcinoma, neuroblastoma, carcinoma of the pancreas, glioblastoma multiforme, cervical cancer, cancer of the stomach, bladder cancer, hepatocellular carcinoma, mammary cancer, colorectal carcinoma, and head and neck cancer, retinoblastoma, cancer of the stomach, blastoma, osteocarcinoma, bone tumor, adult bone malignant fibrous histiocytoma, bone in children malignant fibrous histiocytoma, sarcoma, pediatric sarcomas, nose type natural killer cell, vegetation, plasma cell tumor, myelodysplastic syndrome, neuroblastoma, Testicular Germ Cell Tumors, intraocular melanoma, myelodysplastic syndrome, myeloproliferative disorder/myeloproliferative diseases, synovial sarcoma.In addition, disease comprises urticaria pigmentosa, mastocytosis is diffuse cutaneous mastocytosis such as, solitary mastocytoma in the mankind, and the mastocytoma of dog and some rare hypotypes are as epidermolysis, erythrodermic and distensibility of blood vessel (teleangiectatic) mastocytosis, with the mastocytosis of hematologic disease such as myeloproliferative or myelodysplastic syndrome or acute leukemia, the myeloproliferative disease relevant to mastocytosis, mast cell leukemia and other cancers.Other cancers are also included in the scope of disease, include but not limited to below these: cancer, comprises bladder cancer, bladder transitional cell carcinoma, mammary cancer, colorectal carcinoma, kidney, liver cancer, lung cancer, ovarian cancer, carcinoma of the pancreas, cancer of the stomach, cervical cancer, thyroid carcinoma, carcinoma of testis, especially seminoma of testis and skin carcinoma; Comprise squamous cell carcinoma, gastrointestinal stromal tumor (" GIST "); Lymphatic cells system tumor, comprises leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B cell lymphoma, t cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma and Burketts lymphoma; Medullary system hematopoietic system cancer, comprises acute and chronic myelogenous leukemia and promyelocytic leukemia; The tumour of mesenchyme origin, comprises fibrosarcoma and rhabdosarcoma; Other tumours, comprise melanoma, spermocytoma, teratoma, neuroblastoma and glioma; The tumour of maincenter and peripheral nervous system, comprises astrocytoma, neuroblastoma, glioma and schwannoma; The tumour of mesenchyme origin, comprises fibrosarcoma, rhabdosarcoma and osteosarcoma; And other tumours, comprise melanoma, xeroderma pitmentosum, keratoacanthoma, spermocytoma, follicular carcinoma of thyroid, teratocarcinoma, chemotherapy intractable nonseminoma sexual reproductive cell tumour and Kaposi sarcoma, and any metastasis.
In a preferred embodiment of the invention, cancer is solid tumor.Term " solid tumor " refers in particular to mammary cancer, ovarian cancer, colorectal carcinoma and common GI (stomach and intestine) road cancer, cervical cancer, lung cancer, particularly small cell lung cancer and nonsmall-cell lung cancer, head and neck cancer, bladder cancer, prostate cancer or Kaposi sarcoma.
Pharmaceutical composition described in the present invention and product, test kit or combination preparation can be used for the growth suppressing example knurl, reduce gross tumor volume, prevent the transfer of tumour from sending out and the growth of micrometastasis stove or development.Pharmaceutical composition described in the present invention and product, test kit or combination preparation are specially adapted to the treatment patient of prognosis mala or the tumour of radiotherapy or chemotherapy tolerance.
The present inventor is for test a large amount of different tumor types from clone and the bioptic often kind of tumour of patient (comprising melanoma, glioblastoma multiforme, cancer).Wherein good to the response for the treatment of more than 80%.Particularly, effect is observed to tumor type below: melanoma, glioblastoma multiforme, mammary cancer, colorectal carcinoma, gastrointestinal cancer, liver cancer and head and neck cancer.
In a preferred embodiment, cancer can be selected from melanoma, glioblastoma multiforme, mammary cancer, colorectal carcinoma, gastrointestinal cancer, liver cancer and head and neck cancer.
scheme, dosage and route of administration
The effective dose of each COMBINATION OF THE INVENTION adopted in combination preparation of the present invention can according to adopted particular compound or pharmaceutical composition, method of application, the illness for the treatment of, the severity of illness for the treatment of and different.Therefore, according to many factors, comprise route of administration and patient's states, select the dosage regimen of combination preparation of the present invention.Have the doctor of ordinary skill, clinician or animal doctor can easily determine and the place of opening for preventing, resisting or suppress the significant quantity of single activeconstituents of disease progression.Obtaining the best tolerance range needs in the activity component concentration in generation curative effect in avirulent scope based on activeconstituents in the dynamic (dynamical) scheme of the availability of target site.
Can by identical approach or the Dbait molecule being used endosome solvating agent and combination by two kinds of different approach.Endosome solvating agent, be preferably chloroquine or Oxychloroquine, be more preferably chloroquine, and/or the route of administration of Dbait molecule combined can be oral, parenteral, intravenously, knurl are interior, subcutaneous, encephalic, intra-arterial, locally, rectum, in skin, intracutaneous, intranasal, intramuscular, intraperitoneal, bone etc.In a preferred embodiment, near tumor locus to be treated, use or inject the Dbait molecule of combination.
In a specific embodiment, by oral route or pass through intraperitoneal routes, endosome solvating agent is used preferably by oral route, be preferably chloroquine or Oxychloroquine, be more preferably chloroquine, and intratumor injection can be passed through, by subcutaneous injection, by peritoneal injection, by intracranial injection, by intravenous injection or by oral route, more particularly by knurl, subcutaneous or intravenous injection, also use the Dbait molecule of combination most preferably by subcutaneous route.
In another embodiment, endosome solvating agent, be preferably chloroquine or Oxychloroquine, be more preferably both Dbait molecules of chloroquine and combination all by intratumor injection, by subcutaneous injection, by peritoneal injection, by intravenous injection or pass through oral route, more particularly by knurl, subcutaneous injection or by oral route, also use most preferably by oral route.When by endosome solvating agent, be preferably chloroquine or Oxychloroquine, be more preferably the Dbait molecule of chloroquine and combination inject altogether time, in the limited field of toxicity, endosome solvating agent, the preferably amount of chloroquine are higher, and result for the treatment of is better.The advantage of injection or local injection is altogether, does not need to mate the pharmacokinetic curve in blood plasma.In very concrete embodiment, use endosome solvating agent by oral route, be preferably chloroquine, and used the Dbait molecule of combination by subcutaneous injection.The present inventor confirms, the result of the exception of this route of administration combination provides the pre-treatment of chloroquine, thus establishes the stable state scheme of chloroquine in blood plasma.
Using Dbait molecule or first 2 hours of hairpin nucleic acid molecule that cholesterol combines and/or simultaneously, more preferably using coDbait first 2 hours, using endosome solvating agent, being preferably chloroquine or Oxychloroquine, being more preferably chloroquine.
In the first preferred embodiment, treatment plan comprises step below: before the Dbait molecule starting to combine by cholesterol or hairpin nucleic acid molecule treatment, carry out pre-treatment with endosome solvating agent, preferably chloroquine to patient.Such as, when using (such as topical application) endosome solvating agent near tumor locus to be treated, endosome solvating agent can be used together with the Dbait molecule combined or hairpin nucleic acid molecule, or before the Dbait molecule using combination or hairpin nucleic acid molecule at least or about 1,2,3,4 or 5 hour, preferably between about 1 to 3 hour, more preferably from about 2 hours, use endosome solvating agent.Or, when being used endosome solvating agent by general, can for more time also by longer treatment before the Dbait molecule using combination or hairpin nucleic acid molecule, preferably during the time period in precontract 1 to 3 week of the Dbait molecule or hairpin nucleic acid molecule of using cholesterol combination, during being more preferably the time period of about about 2 weeks, use endosome solvating agent.
Once after using or used the Dbait molecule or hairpin nucleic acid molecule that cholesterol combines, can continue with the process of endosome solvating agent, as long as the Dbait molecule or hairpin nucleic acid molecule that cholesterol combines will be used.Or, also can stop with the process of endosome solvating agent.
When combinationally using DNA damage antineoplastic agent with the Dbait molecule combined and endosome solvating agent, can by identical approach or the Dbait molecule being used DNA damage antineoplastic agent and cholesterol combination by different approach.The route of administration of DNA damage antineoplastic agent can be oral, parenteral, intravenously, knurl are interior, subcutaneous, encephalic, intra-arterial, locally, rectum, through skin, intracutaneous, in intranasal, intramuscular, bone etc.
In a specific embodiment, pass through oral route, simultaneously, separately or sequentially DNA damage antineoplastic agent and endosome solvating agent is used, and can by intratumor injection, by subcutaneous injection, by peritoneal injection, pass through intravenous injection, or pass through oral route, preferably by knurl, subcutaneous or peritoneal injection or by oral route, also more particularly by knurl or subcutaneous, use the Dbait molecule of combination.
In another embodiment, DNA damage antineoplastic agent is used by oral route, and can by intratumor injection, by subcutaneous injection, by peritoneal injection, by intravenous injection or pass through oral route, preferably by knurl, subcutaneous or peritoneal injection or pass through oral route, also more particularly by knurl or subcutaneous, simultaneously, separately or order use Dbait molecule and the endosome solvating agent of combination.
In further embodiment, endosome solvating agent is used by oral route, and can by intratumor injection, by subcutaneous injection, by peritoneal injection, by intravenous injection or pass through oral route, preferably by knurl, subcutaneous or peritoneal injection or pass through oral route, also more particularly by knurl or subcutaneous, simultaneously, separately or order use Dbait molecule and the DNA damage antineoplastic agent of combination.
In other embodiment, by intratumor injection, by subcutaneous injection, by peritoneal injection, by intravenous injection or pass through oral route, preferably by knurl, subcutaneous or peritoneal injection or pass through oral route, also more particularly by knurl or subcutaneous injection, simultaneously, separately or order use the Dbait molecule of DNA damage antineoplastic agent, endosome solvating agent and combination.
In radiation and/or before using DNA damage antineoplastic agent and/or simultaneously and/or afterwards, more preferably in radiation and/or before using DNA damage antineoplastic agent and/or simultaneously, Dbait molecule or the hairpin nucleic acid molecule of endosome solvating agent and combination is used.Carry out radiation and/or use DNA damage antineoplastic agent, condition be when apply radiation or when DNA damage antineoplastic agent arrival tumour cell time, in conjunction with Dbait molecule be present in tumour cell.Based on activeconstituents, activeconstituents at the availability kinetics of target site or the pharmacokinetic curve of activeconstituents in blood plasma, having the doctor of ordinary skill, clinician or animal doctor can determine scheme.PRELIMINARY RESULTS shows, in conjunction with Dbait molecule within the time of one day, keep active.In the first preferred embodiment, treatment plan comprises step below: before starting with the Dbait molecule combined or hairpin nucleic acid molecule treatment, with endosome solvating agent, is preferably chloroquine or Oxychloroquine, is more preferably chloroquine and carries out pre-treatment to patient.Then, when starting to treat with the Dbait molecule combined or hairpin nucleic acid molecule or after treating with the Dbait molecule combined or hairpin nucleic acid molecule, applying radiation or using DNA damage antineoplastic agent.Such as, starting 3-24h after with the Dbait molecular therapy combined, applying radiation or using DNA damage antineoplastic agent.Also can use the Dbait molecule of DNA damage antineoplastic agent and combination simultaneously.
Once begin through radiotherapy or after treating with DNA damage antineoplastic agent, the Dbait molecular therapy with endosome solvating agent and/or combination can be continued, as long as will apply or use by radiotherapy or the treatment carried out with DNA damage antineoplastic agent.Or, also can stop the Dbait molecular therapy with endosome solvating agent and/or combination.
For the Dbait molecule combined, the effective dose of the DNA damage antineoplastic agent adopted in combination preparation of the present invention, test kit or product may according to method of application, the illness for the treatment of, the severity of illness for the treatment of and different.Therefore, select the dosage regimen of the Dbait molecule combined according to many factors, described factor comprises route of administration and patient's states.Have the doctor of ordinary skill, clinician or animal doctor can easily determine and the place of opening for preventing, resisting or suppress cancer progression, particularly treat the significant quantity of the Dbait molecule of combined combination with selected DNA damage.
Such as, for topical application (such as, when use in knurl or subcutaneous administration time), in conjunction with the significant quantity of Dbait molecule be at least 0.01mg/1cm 3tumour, preferred 0.1-40mg/1cm 3tumour, most preferably 1-20mg/1cm 3tumour.Described significant quantity can be used with treatment plan every day (such as, 5 days weekly, continue 3 to 6 continuous weeks, or one week 3 times, continue 3 to 6 all continuously).Or, such as, can use at least 0.01mg/1cm with the weekly treatment scheme in lasting 3-6 continuous week 3tumour, preferably 0.1-40mg/1cm 3tumour, most preferably 1-20mg/1cm 3the significant quantity of tumour.When using other route of administration, those skilled in the art can adjust described amount, thus to obtain the significant quantity of Dbait molecule in tumour combined be at least 0.01mg/1cm 3tumour, preferred 0.1-40mg/1cm 3tumour, most preferably 1-20mg/1cm 3tumour, particularly every day treatment plan or weekly treatment scheme in.Such as, for systemic routes, in conjunction with the significant quantity of Dbait molecule or unitary dose can be 0.1 to 100mg, be preferably 4 to 40mg.Therefore, for systemic routes, in conjunction with the significant quantity of Dbait molecule or unitary dose can be 0.06 to 0.6mg/kg patient.Certainly, consider chemotherapy and/or radiotherapeutic scheme, those skilled in the art can adjust dosage and scheme.
For endosome solvating agent, particularly chloroquine or Oxychloroquine, be more preferably chloroquine, the effective dose of the endosome solvating agent adopted in combination preparation of the present invention, test kit or product may according to method of application, the illness for the treatment of, the severity of illness for the treatment of and different.Therefore, select the dosage regimen of endosome solvating agent according to many factors, described factor comprises route of administration and patient's states.Have the doctor of ordinary skill, clinician or animal doctor can easily determine and the place of opening for preventing, resisting or suppress cancer progression, particularly treat the significant quantity of combined endosome solvating agent with the Dbait molecule combined and selected DNA damage.
In a specific embodiment, and if when using the known selected endosome solvating agent of oral route to can be used for treatment or prevention of malaria, use endosome solvating agent, particularly chloroquine or Oxychloroquine for treatment or the same dose of prevention of malaria and scheme, be more preferably chloroquine.Such as, if selected endosome solvating agent is chloroquine or Oxychloroquine, be more preferably chloroquine, then with 100-600mg/ days, preferably 200-400mg/ days, more preferably from about 300mg/ days, weekly, chloroquine or Oxychloroquine can be used twice, three times or four times.In a specific embodiment, to use chloroquine or Oxychloroquine over about 100mg/ days during 1 or 2 week, or biweekly chloroquine or Oxychloroquine can be used with about 300mg during 1 or 2 week.
In another embodiment, when contemplating approach in topic route such as subcutaneous or knurl, endosome solvating agent, particularly chloroquine or Oxychloroquine can be used with 100-300mg, being more preferably chloroquine.
For radiotherapy, any radiation therapy plan known in the art can be used, particularly stereotaxis radiation (such as 15Gy) or gradation radiation (fractionated radiation).Use gradation radiation may be effective especially, such as, can within the time of 1,2,3,4,5 or 6 week, every day or every 2-5 days, preferably every 3-4 days apply radiation.Radiation can be 1 to 10Gy, preferably 2 to 5Gy, particularly 2,3,4 or 5Gy.Such as, it is contemplated that the gradation radiation of 15x2Gy in 6 weeks, or the gradation radiation of 4 to 6x5Gy in 2 weeks.In a preferred embodiment, contemplated radiotherapy is the scheme of interior 4 5Gy radiation at 2 weeks.Test the different schemes or condition of using radiation and Dbait molecular combinations Therapeutic cancer, and allowed to prove that the radiation sensitization of Dbait molecule to tumour depends on the dosage of Dbait molecule, instead of radiation dose.
For chemotherapy, adopt in combination preparation of the present invention, test kit or product or may according to adopted concrete DNA damage antineoplastic agent, method of application, the illness for the treatment of, the severity of illness for the treatment of and different with the effective dose of the DNA damage antineoplastic agent of combination of compositions of the present invention.Therefore, select the dosage regimen of DNA damage antineoplastic agent according to many factors, described factor comprises route of administration and patient's states.Have the doctor of ordinary skill, clinician or animal doctor can easily determine and the place of opening for preventing, resisting or suppress the significant quantity of DNA damage antineoplastic agent of cancer progression.
Described treatment can comprise one or several circulation, such as 2 to 10 circulations, particularly 2,3,4 or 5 circulations.Described circulation can be lasting or separate.Such as, respectively circulate by 1 to 8 week, preferably the time period in 3 to 4 weeks separates.
Experimental section below can disclose other aspects of the present invention and advantage, and described experimental section should be considered to illustrative, and does not limit the scope of the application.Refer to many reference in this specification sheets, in these reference quoted, each is all by reference to being incorporated into this.
Embodiment
With polymine (PEI) compound or the distribution of short dna (Dbait) be combined with cholesterol and active multiple dimensioned comparison.
The sign of Dbait/ carrier complexes and cellular uptake
Show, PEI can and DNA, oligonucleotide and RNA form mixture between non-covalent polyelectrolyte.Long PEI chain is highly effective in gene transfection, but has more cytotoxicity.The present inventor tests several PEI particle polymer with Dbait, and is compared by the Dbait (being called coDbait) of their activity with the modification being covalently attached to cholesterol.CoDbait is covalently bound Dbait molecule with the aliphatic chain of cholesterol, can use coDbait without the need to other carrier.For tested each carrier, the major objective of the present inventor develops the preparation under the highest Dbait concentration with the most uniform size distribution.Diameter and the surface charge of particle is measured by Dynamic laser scattering (DLS).Utilize multimodal analysis, the present inventor finds that mean sizes is that the PEI (bPEI25K) of the branching of 25Kd and linear PEI and the Dbait that is of a size of 22Kd (PEI22K) or 11Kd (PEI11K) form the mixture (table 1) with similar characteristics.
The fluorescence of the Dbait that table 1. is prepared and cellular uptake
afluorescence under FL2 value; bmCC=average cell content F L2 value (>3 experiment); cthe cell content FL2/ fluorescence that cor MCC=corrects
Test the different ratios of PEI to Dbait.The lowest ratio causing 100%Dbait compound is determined by gel shift test.For PEI11K, PEI22K and bPEI25K for further study have selected the N/P ratio of 6,6 and 9 respectively.In 10% sucrose, Dbait-PEI composite particles was stable 1 hour period.The form (size range is 125 to 140nm) (Figure 1A) of the high uniformity of spheroidal particle in colony is confirmed by transmission electron microscope.In dilution buffer, there is concentration more than the salt of 0.8mg/mL or long storage time, PEI mixture can be caused to assemble.The Superfect mixture (60 μ gSuperfect/ μ g Dbait) (>2 μm) producing the aggregate of larger and polymolecularity is used as positive control.Uncharged amphipathic multipolymer Lutrol does not form stable interactional mixture with Dbait, and is used as negative control in some experiments.
The Dbait that the present inventor uses fluorescence cy3-to modify is to monitor the cellular uptake of different composite thing.The initial fluorescence of cy3-Dbait mixture is monitored immediately before transfection.In PEI mixture, Dbait fluorescence reduces by 2 to 3 times, and this shows that the tight fasciculation of this molecule and PEI may quenching fluorescence (table 1).The fluorescence of coDbai is also low than naked Dbait, and this shows that cholesterol may interact on the same molecule with cyanine.Superfect or Lutrol does not affect fluorescence.Fibroblastic cell content of people's transfection is measured by flow cytometry.Do not have different by the fluorescence distribution of the cell after naked Dbait or Dbait-Lutrol mixture process from undressed contrast, this shows that Dbait molecule does not have spontaneously to enter into cell.Electroporation is relative inefficiencies, and the concentration increasing Dbait does not improve transfection efficiency.All polycationic polymers (PEI and superfect) all promote effective cellular uptake, but linear PEI demonstrates the distribution wider than Dbait/superfect or Dbait/PEIb25K mixture.CoDbait enters cell under the help not having transfection agents, but efficiency ratio Dbait/PEI is low 10 times.Make coDbait concentration increase 10-15 and doubly allow effective transfection (Figure 1B).
A restriction of DNA transfer efficiency is that it is retained in endosome, which prevent it and its target interacts, or prevents it transcribed.Enter in cell, DNA must escape from normal endosome approach, and described normal endosome approach can cause degraded.Therefore, the delivery efficiency of DNA is not only relevant to cellular uptake, but also to destabilization with escape relevant from endosome.Known PEI has high surge capability, and high surge capability is conducive to DNA from endosome and lysosome release (" proton sponge hypothesis ").On the contrary, coDbait needs the help of film fusogen such as chloroquine (CQ), effectively to discharge from endosome.From the angle improving transfection efficiency, we with the addition of 100 μMs of CQ to cell half an hour before transfection.CQ makes the cellular uptake of coDbait improve 2-4 doubly (Fig. 1).The amount (Quanz etc., 2009, the same) of the Dbait be released in cell is monitored by be combined the kinase whose activation of triggered DNA-PKcs with Dbait molecule by DNA-PKcs kinases.Add cholesterol and do not affect the ability (Fig. 2 A) that Dbait activates the DNA-PK of purifying.In cell, carry out the kinase whose activation of monitoring of DNA-PKcs by the amount of the H2AX phosphorylation strictly depending on DNA-PK according to the show.H2AX phosphorylation (Fig. 2 B) is all induced in Dbait/PEI and coDbait cell after treatment.Branching and linear PEI/Dbait mixture impel H2AX phosphorylation (Fig. 2 C) fast, and H2AX phosphorylation reaches the highest in 6 hours after beginning transfection, and continues 24 hours after transfection.After electroporation, the kinase activity of Dbait induction is all low-down (Fig. 2) at any time.High density CoDbait is unusual poor efficiency for the H2AX of phosphorylation, and needs to reach maximum value at least 24 hours.During transfection, add CQ makes the DNA-PKcs in coDbait transfectional cell activate the viewed level of Dbait/PEI (Fig. 2 C) be increased to few 10 times.CQ does not make the activity in Dbait/PEI transfectional cell increase, and this shows that Dbait is discharged from endosome effectively when Dbait and PEI compound tense.Due to after transfection between 5 hours and 24 hours, the cellular uptake of coDbait does not increase, and therefore, the slow activation of coDbait to DNA-PK shows that it discharges from endosome lentamente.
Cellular uptake in zebra fish body early embryo and overall toxicity
Cell culture is analyzed Dbait picked-up do not allow to sum up about the drug diffusion in whole organism, cellular uptake and activity with activity.The present inventor assesses this problem (Kimmel etc., 1995, Dev Dyn 203:253-310) by being expelled in the lacuna of 1000 cell stages (1K phase) zebrafish embryo by naked Dbait-cy3 or the Dbait-cy3 with adjuvant.The program allows to carry out observing in body to the activity of Dbait-cy3 on the quick somatoblast of the distribution of cell and subcellsular level and zebrafish embryo in early days thereof by Laser Scanning Confocal Microscope.The naked Dbait-cy3 being injected at the animal pole place of 1K phase embryo diffuses through rapidly whole blastodisc, and within 15 minutes, just no longer can detect after injection.Add Lutrol to allow Dbait to be retained in the space, extracellular around injection point, but do not promote cellular uptake.Under the condition that there is Superfect or PEI, in cell, observe many fluorescence patches, this shows effective cellular uptake.CoDbait cy3 demonstrates the behavior of another kind of type, and the behavior has strong and lasting plasmalemma dyeing and patch shape intracellular Fluorescence.Before the injection embryo and CQ are hatched, make large coDbait fluorescence patch be transformed into distribution in dispersivity cell.
Within 20 hours, observing phenotypic effect after injection allows overall toxicity that is active to Dbait and treatment to assess.In the cell head of the young, Dbait fluorescence (Fig. 3 A-C) within 24 hours, detected after injection, fate map according to zebra fish grows (Woo etc., 1995, Curr Opin GenetDev 5:439-443), described cell head stems from the injection areas at the animal pole place of embryo before gastrula.Inject without the Dbait of adjuvant (NA) or demonstrate growing affecting (Fig. 3 D) with the Dbait that Lutrol (Lu) combines, this is relevant to the weak cellular uptake of above-mentioned Dbait.Add adjuvant and cause the necrocytosis in head and relevant to volume injected, a large amount of necrocytosiss and teratogenesis may be observed.As described, 24 hours phenotypes are classified (Fig. 3), allow to carry out quantitatively the toxicity of injection mixture.For the Dbait of same concentrations, according to the difference of adjuvant, in necrocytosis and heteroplasia subsequently, there is obvious difference.It is very poisonous for adding Superfect (sup) to embryonic cell, and early stage a large amount of necrocytosiss cause a high proportion of 2 type phenotypes.Similarly, add PEI (25K, 22K, 11K) and be proved to be toxic to zebra fish blastomere.Although it is low that efficiency ratio adds PEI, injection coDbait causes significant necrocytosis.Embryo and CQ preincubate are not significantly increased toxicity.In a word, body early embryo necrocytosis and heteroplasia are subsequently for assessment of the quick of the overall toxicity of Dbait+/-adjuvant in zebrafish embryo and reliable scheme.The dependency of necrocytosis and cellular uptake shows, the anti-tumor activity of Dbait in embryonic cell may play important effect in toxic effect.
Local in mouse and general toxicity
In order to assess the consistence of cell culture, zebrafish embryo and mouse data, the present inventor analyzes the tolerance of nude mice skin to repetitive administration Dbait/PEI1K, Dbait/PEI22K, Dbait/bPEI25K and coDbait.After 3 every day subcutaneous (SC) injections, analyze the toxicity of the Dbait of different preparations.All Dbait/PEI all demonstrate high toxicity, wherein tolerate the injection of 3.75mg/kg, but from 5mg/kg, injection triggers the local inflammation relevant to local necrosis and ischemic, and after stopping the treatment, local necrosis and ischemic rapidly disappear.Intravenously (IV) is injected toxicity and is provided similar result: the Dbait/PEI intravenous injection of 3mg/kg is lethality, and the death wherein occurred in injection process may be blocked by blood and cause.Make tolerance bring up to 6mg/kg Dbait/PEI (6 nmoles/injection) by pouring into slow injection (0.4 μ L/mn), this confirms that most of IV toxicity is that partial concn owing to injecting position causes.No matter employ which kind of approach: SC, IV inject or IV perfusion, use or under tested all dosage (up to 800mg/kg/ injection, 800 nmoles/injection), all do not demonstrate any toxicity with the coDbait of CQ.
Anti-tumor activity in xenograft tumor
Combine with radiotherapy, the heteroplastic Humanmachine tumour of SK28 tests the anti-tumour effect of the Dbait of preparation.In each radiation first 5 hours, utilize intratumor injection to use Dbait/ carrier complexes.
Carry out drug administration although used in many tests by intratumor injection (IT), suggestion at present should avoid this route of delivery in clinical trial.Present inventors studied and how can use Dbait/PEI11k or coDbait by subcutaneous injection in tumor vicinity region (SC).Several clinical trial successfully employs this route of administration.First the present inventor compares the molecular diffusion (Fig. 4) in the tumour after with 1 intratumor injection or twice subcutaneous injection process performing at the opposite side of tumour.Tend to form aggregate in injection site with the fluorescence Dbait of PEI11k compound, and be progressively diffused into the edge of tumour.On the contrary, around injection, be no matter in inside tumor or in its vicinity, coDbait all demonstrate evenly distribution.In tumor growth control, SC injects Dbait/PEI11k or coDbait and injects lower slightly effect (table 2) than IT.But the number increasing injection site will allow significantly to improve tumor growth and control, and does not increase local toxicity.
Table 2. is in the survival of radiation from xenograft mouse after various different therapeutic combination
amethod of application: IT, in knurl; SC, subcutaneous
bcure the animal that mouse is nothing recurrence in 300 days after the treatment
ctGD calculates and statistical analysis is described in materials and methods
Have studied the survival that 5 groups carry the nude mice of SK28 melanoma xenograft.Group 1) do not treat mouse (n=16); Group 2) radiation murine (IR, n=12); Group 3) radiation murine (CQ, IR, n=10) of peritoneal injection 1mg chloroquine; Group 4) carry out the treatment mouse (DT01 of radiation after 5h by intratumor injection 0.6mgDT01 (also referred to as coDbait), IR, n=11), and group 5) peritoneal injection 1mg chloroquine, intratumor injection 0.6mg DT01 (also referred to as voDbait) carry out the pretreatment of mice (DT01 of radiation after 5h after 2 hours, CQ, IR, n=13).
Result is shown in Figure 5.
Compared with independent radiotherapy (organizing 2), along with using 0.6mg coDbait in knurl, the remarkable radiation sensitization of the pre-treatment undertaken by chloroquine also makes survival improve (group 5), and coDbait (group 4) or CQ (group 3) does not demonstrate significant radiation sensitization.Degree after the radiation sensitization degree 0.06mgDbait be similar to by prepare with N/P ratio=6 with polymine (PEI) of group 5 treats.
On the nude mice carrying SK28 melanoma xenograft, also have evaluated the application program based on subcutaneous injection coDbait.The program is schematically disclosed in figure 6.Briefly, the program comprises coDbait and the 4 kind combined therapies of radiation in two weeks.Particularly, at two relative some subcutaneous injection 4mg coDbait, described two relative points and tumor boundaries are separated by 5mm.In addition, with 1mg chloroquine (CQ), pre-treatment is carried out to animal, and process with same dose with CQ further during with coDbait and radiation therapy.At the later evaluation tumor growth of this application program, in Fig. 6, give result.
Observe, after with chloroquine pre-treatment, during with coDbait and radiation and chloroquine co-processing, observed minimum tumor growth.In addition, with the group of chloroquine co-processing demonstrate than without chloroquine process those groups evenly result.
Conclusion
In this research, inventors used series of experiments to guide the exploitation of application program and pharmaceutical preparation.The different preparations of Dbait are compared in these tests before making it possible to carry out preclinical test on mouse.Cell and zebrafish embryo test are used to the efficiency assessing Dbait cellular uptake, and select most suitable scheme and preparation for the preclinical study on Mammals, and described cellular uptake is the prerequisite steps in antitumor drug effect.Overall toxicity in zebrafish embryo does not have dependency with the toxicity in mouse skin or the toxicity after systemic injection.Particularly, the high toxicity of coDbait in zebrafish embryo shows that great majority touch the cell possibility death of medicine, and mouse skin does not demonstrate any reaction to the injection of coDbait high dosage.This difference shows, the toxicity in zebra fish body early embryo is the index of tumor sensitivity instead of health tissues susceptibility.In fact, according to the show, Dbait molecular specificity ground is toxic in tumour, but in normal skin nontoxicity (Quanz etc., 2009, the same).Dbait/PEI (5 μMs) and coDbait (50 μMs)+CQ trigger suitable DNA-PKcs and activate in cell culture, zebrafish embryo has similar toxic effect (Fig. 3 D), and on mouse tumor, demonstrate significant anti-tumor activity (table 2, Fig. 5 and 6).This observations is consistent with the susceptibility of the anti-tumor activity of zebrafish embryo cell, and described zebrafish embryo cell and tumour cell have common characteristic attributes, comprises mitotic index and biochemical and phenotypic characteristic.Consistent with this hypothesis, the present inventor more recently by by direct for naked Dbait endocellular injection in the zebra fish blastomere between 1 and 16 cell stages, confirm the antiproliferative activity of Dbait.
PEI polymkeric substance is the most effective in formation Dbait mixture in tested all adjuvant molecules.But their toxicity organizationally and in blood system limits their application.By slowly using (perfusion) and by injected dose is dividing in different injection sites, partly overcoming local toxicity.But the combination of the covalency of cholesterol and Dbait provides for Dbait being delivered to cell and optimum substituent without the need to adding adjuvant.In fact, in tested dosage range, there is not toxicity, show that this molecule may prove useful, although anti-tumour effect needs certain maximum dose level.The dosage that Dbait/PEI11K and coDbait is respectively 3 nmole/injections and 30 nmole/injections makes the tumor growth delay caused separately by radiation double.The relative ratios that these two kinds of preparations of Dbait/PEI11K and coDbait (6 nmoles and >800 nmole) respective toxicity provides effective dose/toxicity dose is respectively 0.5 and <0.037, and this shows that coDbait is an extraordinary clinical trial material standed for.
Materials and methods
dbait and particle are formed
Dbait and coDbait molecule is from Eurogentec (Seraing by automatic solid phase oligonucleotide synthesis, Belgium) or from Agilent Technologies Nucleic Acid SolutionDivision (Boulder, USA) obtain, (Quanz etc. as previously mentioned, 2009, the same).By sex change reversed-phase HPLC and/or HPLC-IEX, purifying is carried out to them.Some Dbait derivative is marked with fluorophor Cy3 (λ excite=540nm, λ launch=560nm) or Cy5.5 (λ swash send out=X nm, λ launch=X nm).Linear PEI (11kDa and 22kDa) comes from Polyplus-Transfection (Illkirch, France), and is provided as the instant solution of 300mM nitrogen concentration.The bPEI25kd of branching is purchased from SIGMA-Aldrich (Saint Quentin, France).Lutrol is purchased from In Cell Art (Nantes, France).Dbait and PEI solution (PEI stoste) is diluted in 10% sucrose or 150mM NaCl (for in-vitro transfection experiment), to obtain different carriers/Dbait ratio.According to the amine nitrogen number of PEI and the phosphoric acid salt number of Dbait, determine the ratio (or N/P ratio) of PEI/Dbait.Usually, for the mixture of 300 μ L under 0.6mg/mL and N/P 6, by Dbait (180 μ g, 0.54 μm of ol phosphoric acid salt) and the polymers soln (11, the 4 μ L PEI stostes containing 0.3 μm of ol amine nitrogen) of aequum be diluted to 150 μ L (10% sucrose) separately.According to manufacturers (Qiagen, Courtaboeuf, France), prepare Superfect/Dbait particle with the ratio of 10 μ l Superfect/ μ g DNA.The compound of carrier/Dbait is analyzed by agarose gel electrophoresis method for detecting.Mixed with Bromophenol Blue dye (1 μ L) by sample (18 μ L), then load is on 1,5% sepharose in the electrophoresis chamber containing TAE damping fluid 1X (40mM Tris-acetate, pH 8.3,1mM EDTA).Gel is run 30 minutes under 100 volts.Then use ethidium bromide (EtBr) by gel-colored 15 minutes, and observe band under w light.
cell cultures, Dbait molecule and transfection
Dbait molecule is prepared by automatic solid phase oligonucleotide synthesis.Sequence is 5'-GCTGTGCCCACAACCCAGCAAACAAGCCTAGA-(H)-TCTAGGCTTG TTTGCTGGGTTGTGGGCACAGC (SEQ ID No 4), and wherein H is six ethylene glycol joints.Research during the inoblast MRC-5 using SV40 to transform cultivates on cell.At 100% humidity, 95% air and 5%CO 2condition under, cell grows in the complete DMEM (Gibco, Cergy Pontoise, France) with 10%FCS and microbiotic (100 μ g/mL Streptomycin sulphates and 100 μ g/mL penicillin) with the form of monolayer culture at 37 DEG C.Unless otherwise stated, carry out transfection in the MEM substratum of 1.2mL serum-free in the plate of 60mm diameter.According to the specification sheets of manufacturers, be carry out transfection with jetPEI (Polyplus-transfection, Illkirch, France) 6 times at N/P ratio.Briefly, Dbait to be diluted in 150mM NaCl and softly to mix with the equal-volume PEI in 150mM NaCl, and joining in the DMEM substratum of serum-free.CoDbait is introduced directly in the DMEM substratum of serum-free.If do not illustrated in addition, in the DMEM substratum (in the plate of 60mm diameter) of 1.2ml containing serum, carry out the Dbait molecule transfection of 5 hours with Superfect reagent, then allow cellular-restoring 1 hour.For electroporation, use Gene Pulser II (Bio-Rad, Marnes-la-Coquette, France) 2 μ g Dbait transfections 1.2 × 10 6individual cell.At the end of 5 h transfection (zero-time), substratum is replaced by perfect medium, and by the time specified by Growth of Cells, then analyzes.Chloroquine (50 μMs) within first 30 minutes, is added in transfection.
flow cytometry
With the different mixture transfectional cell 5h with Dbait-cy3, and allow Growth of Cells 5 hours or 24 hours, wash with PBS immediately.By flow cytometry direct analysis cell.For the Immunofluorescence test undertaken by flow cytometry, cell is fixed 10 minutes in 2% paraformaldehyde, then carry out immunodetection.Note, saturatingization process eliminates most of Dbait, weakens the Immunofluorescence test on same cell and Dbait detection.Cell is fixed 15 minutes in 4% paraformaldehyde, saturatingization 1 hour in 0.2%Triton X-100, close with 2%BSA, and with the mouse monoclonal antibody (UpstateBiotechnology of the anti-γ of first antibody-H2AX, Temecula, CA, USA) 2 hours are hatched on ice, and with being combined with Alexa-488 (Molecular Probes, Eugene, OR, USA), the red (Rockland of Texas, Gilbertsville, PA, USA) second antibody at room temperature appear 30 minutes with 1/200 dilution.By cells rinsed with PBS, and be resuspended in the PBS with 50 μ g/mL propidium iodides, 25U/ml RNaseA.By FACScalibur flow cytometer (BDBiosciences, Franklin Lakes, NJ, USA) analysis of cells, and use BD CellQuestPro (BD Biosciences) and free WinMDI 2.8 (Scripps Research Institute, La Jolla, CA, USA) software analysis data.
the raising of zebra fish, embryo collection and process
Zebra fish-egg is obtained from the natural spawning of wild-type or transgenosis (β actin:egfp-ras) fish products kind.Be fixed on the Dbait injection that Narishige (MN 153) micromanipulator having to fall to penetrating on the Anatomical observation instrument (dissectting scope) of fluorescent lighting (Leica MZ16F) and air syringe (Eppendorf FemtoJet) is used to perform 1K cell stage.Instrument (KopF vertical pipette puller) (KopF 720) drawn glass kapillary (Harvard Apparatus GC100-10) is drawn, to make entry needle with the vertical transfer pipet of KopF.By the animal pole place of the embryo of Dbait injection of solution in the cell cycle 10 of 2 to 5nl, and process immediately, to carry out laser scanning co-focusing micro-imaging (erect type Leica SP2) by the lens objectives of 40x/0.8NA water soaking.Excited by 480nm (eGFP) and 561nm (cy3) simultaneously, carry out imaging.By embryo at 28.5 DEG C further growth to 24hpf.Observe the young of an age in days dissecting under station (dissecting stop), and as shown in Figure 3 phenotype to be classified.Chloroquine before injection is treated to hatches with 50 μMs of chloroquines 2 hours (zebra fish handbook (The Zebrafish Book)) in embryo culture medium.Separately or combine injection 50 μMs of Dbait (coDbait)-cy3 with PEI25K (N/P ratio=9), PEI11K (N/P ratio=6), Superfect (10 μ l/1 μ g Dbait).
dbait in mouse and radiation therapy
By by 10 6individual tumor cell injection is to Adult female nude mice (Charles River strain; L ' arbresle, France) oxter, obtain SK28 or U87G xenograft tumours.In at least one week before entry into the trial, animal is settled in the lab.Under the controlled condition of light and shade circulation (12 hours: 12 hours), relative humidity (55%) and temperature (21 DEG C), every cage has 5-6 animal.Can freely obtain food and tap water.After about 12 days, be 150-200mm when measuring Subcutaneous tumor 3time, divided by mouse uniformly group to accept different treatments, often to organize 12 mouse at the most.With 137cs unit (0.5Gy/min) carries out radiation, and the guard shield adopted is designed to about 2/3rds of the health that watches for animals.By thermoluminescence dosimetry control dose.During 2 weeks, every 3 phase 5Gy/ weeks, divided for 6 phases sent the total dose of 30Gy.As above described by vitro study, be prepared in by Dbait molecule in 100 μ L 10% sucrose, difference is without NaCl to perform PEI mixture (Polyplus Transfection, Strasbourg, France).Before the injection, Dbait mixture is at room temperature hatched 15min.With required concentration, CoDbait is diluted in 10% sucrose.In each radiotherapy phase first 5 hours, carry out the intratumor injection of the Dbait of specified amount.According to the scheme of correlation test, with 100 μ L 10% glucose, the animal that Mock treats is injected.Within every 3 days, estimate tumor size by vernier caliper measurement, and through type (2x length x width 2) carry out calculated size.Mouse is weighed, and takes weekly the photo of tumour.For ethical reasons, when the tumour of animal reaches 2000mm 3time, they are put to death.The terminal used in survival analysis is dead day.The local Animal Experimental Ethical council (Local Committee on Ethics of AnimalExperimentation) (Orsay, France) have approved all experiments.
statistical analysis
For often kind for the treatment of and often kind of tumor type carry out the descriptive analysis of tumor response.Within 1st day, be first and treat same day phase.All tracked at least 150 days of all animals.Median life is estimated according to Kaplan-Meier method.Deducted the time of the mean tumour volume four times of control group by the time of the gross tumor volume four times of single mouse from each treatment group, calculate tumor growth delay (TGD).Each measuring result is used to be that each treatment group calculates TGD mean value.Estimate to assess total survivorship curve by Kaplan-Meier, and compare with distribution free LogRank inspection, because data not followed normal distribution distribution.Analyze with statEL software (ad Science, Paris, France).First total LogRank is carried out to each group with identical tumor type.Then contrasting of the treatment of employing Dbait and mock being treated compares.Size of animal (n), relative risk (RR) and p value are reported in table 2.All tests are all considered to significance under 0.05 significance level.
the physicochemical property of the Dbait particle of preparation
Employing specification is below at Zetasizer nano series (Malvern instruments, Paris, France) particle diameter of carrier/Dbait is measured on by dynamic light scattering (DLS): dielectric viscosity: 1.150cP, specific refractory power: 1.45, scattering angle: 90 °, temperature: 25 DEG C.Data are the mean value that each sample is measured for 3-5 time, and wherein each measurement is averaged to the data of 10-15 Asian Games row.Use the multimodal number distribution software (multimodal numberdistribution software) provided with instrument to data analysis.Zeta-potential is measured, by dilute particles in 10% sucrose/10mM NaCl to obtain the final concentration of 0.1mg/mL, and to measure with specification below: 3 measurements, dielectric viscosity: 1.054cP, the specific inductivity of medium: 79, temperature: 25 DEG C.
aobserve weight ratio during maximum Dbait activity
bby the mean diameter (+/-SD) that dynamic light scattering (see supplementary material and method) measures.
cparticle in 1% sucrose, 10mM NaCl
transmission electron microscope
By carrying out the sample for the preparation of transmission electron microscope with uranyl acetate negative staining.A sample (10 μ L) is deposited on a year net (the formvar/ carbon on 200 order copper, AGAR scientific) go up and place 3 minutes, then removes excessive liquid with blotting paper.Then, with 10 μ L aqueous uranyl acetate (2%), mixture is dyeed 2 minutes, and remove excessive described material with blotting paper.Observe by Jeol JEM-100S Electronic Speculum.
The Dbait molecule of optional combination
Prepare and described below the optional Dbait molecule combined:
The molecule of the combination of following formula (IIe)
wherein
The molecule of the combination of following formula (Ie)
wherein
Suppress by DNA PK the activity having determined the Dbait molecules that these optionally combine, as above describe in detail (Fig. 7).Observe these molecules combined and maintain its activity.Particularly, 5' end or in ring in conjunction with various lipid and part, the capacity these molecules being triggered to DNA-PK activity is less.
In addition, also by measuring the amount of H2AX phosphorylation, with or in clone, do not determine their activity with chloroquine, as above describe in detail (Fig. 8).First, for the Dbait molecule of tested combination, observe when carrying out pre-treatment with chloroquine, their activity is higher.In addition, it may be noted that compared with being combined in hairpin loop with by cholesterol, cholesterol is attached to 5' end, causes more effective molecule (see 0902, comparing with 0815 with 0813) astoundingly.
In conjunction with the cellular uptake of Dbait molecule
The present inventor determines the cellular uptake of the Dbait, the particularly CoDbait that are combined with cholesterol by flow cytometry, and compares with Dbait.
The results are shown in table below.
Start with as table described in various transfection conditions process after 5 hours, carry out the flow cytometry of the cellular uptake in MRC5 clone.By all oligonucleotide of cyanine 3 (Cy3) dye marker: Dbait (Dbait-Cy3), cholesterol-Dbait (0813) (CoDbait-Cy3) and siRNA (the Co_siRNA_H2AX:Cy3-5'-CAACAAGAAGACGCGAAUCTT-3'-cholesterol (SEQ D No 6) of target H2AX at 5' and 3' of sense strand with cyanine 3 and cholesterol; 5'-GAUUCGCGUCUUCUUGUUGTT-3'(SEQ ID No7).When pointing out, before transfection, add 50 μMs of chloroquines (CQ).

Claims (4)

1. the nucleic acid molecule combined, it has one of following formula:
Nucleotide wherein with underscore refers to the Nucleotide being with or without thiophosphatephosphorothioate or methylphosphonate backbone, the L' connected is selected from two (the phosphorus)-8-hydraza-1-hydroxyl-4-oxa--9-oxo-nonadecane of six ethylene glycol, four deoxythymidines (T4) and 2,19-; M is 1 and L is formamido-oligoethylene glycol, and C is selected from dioleoyl, octadecyl, folic acid, tocopherol and cholesterol.
2. the nucleic acid molecule of the combination of claim 1, wherein promotes that the molecule of endocytosis is selected from tocopherol and cholesterol.
3. the nucleic acid molecule of the combination of claim 1, wherein said molecule is
Nucleotide wherein with underscore refers to the Nucleotide with phosphorothioate backbone.
4. pharmaceutical composition, it comprises the nucleic acid molecule of the combination of any one of claim 1-3.
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